BODY SIZE VARIATION IN RANA TEMPORARIA POPULATIONS INHABITING EXTREME ENVIRONMENTS

Cogalniceanu Dan

Abstract: We studied the variation in body size in populations of a widespread anuran species, Rana temporaria, from high altitude and latitudes. Our results indicated a variable interannual pattern of body size, suggesting that body size in extreme environments is influenced by many factors. This indicates that long-term series of observations are needed to separate natural fluctuations from man-induced changes.

Analele Universităţii Ovidius Seria: BIOLOGIE – ECOLOGIE Volumul 14, anul 2010 VOLUM OMAGIAL Ovidius University Annals BIOLOGY – ECOLOGY Series Volume 14, year 2010 OVIDIUS UNIVERSITY PRESS Analele Universităţii Ovidius, Seria Biologie – Ecologie Volumul 14 (2010) VOLUM OMAGIAL (dedicat împlinirii a 20 de ani de la înfiinţarea Facultăţii de Ştiinţe ale Naturii şi Ştiinţe Agricole) Redactor Şef Prof. univ. dr. Marian Traian GOMOIU Membru corespondent al Academiei Române mtg@datanet.ro Redactori Conf. univ. dr. Marius FĂGĂRAŞ fagaras_marius@yahoo.com Prof. univ. dr. Rodica BERCU rodicabercu@yahoo.com Mail address: Faculty of Natural and Agricultural Sciences, “Ovidius” University of Constanţa, Aleea Universităţii nr. 1, corp B, Constanţa RO-900470, România, Tel. 0241605060/ Fax: 0241606432, contact@stiintele-naturii.ro. ORDERING INFORMATION Ovidius University Annals of Biology – Ecology is published annually by Ovidius University Press. The journal may be obtained on exchange basis with similar Romanian or foreign institutions. No part of its publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronically, mechanical, photocopying, recording or otherwise, without the written permission of the Publisher, Ovidius University Press, Blvd. Mamaia 124, RO-900527, Constanţa, România. © 2010 Ovidius University Press ISSN 1453–1267 Ovidius University Annals of Natural Sciences, Biology – Ecology Series, Volume 14 (2010) Contents Limitative mycotic factors for some plants from the Bulgarian coast of the Black Sea Gavril NEGREAN………………………………………………………………………................................ 3 The medicinal plants of Provadiisko Plateau Dimcho ZAHARIEV, Desislav DIMITROV………………………………………………………………... 17 The plants with protection statute, endemites and relicts of the Shumensko Plateau Dimcho ZAHARIEV, Elka RADOSLAVOVA……………………………………………………………... 25 A characteristic of model habitats in south Dobrudja Dimcho ZAHARIEV………………………………………………………………………..……………….. 33 Floristic aspects of the Hills of Camena village (Tulcea county) Marius FĂGĂRAŞ............................................................................................................................................ 45 Identification of some rose genitors with resistance to the pathogens agents attack Marioara TRANDAFIRESCU, Corina GAVĂT, Iulian TRANDAFIRESCU, Elena DOROFTEI ………... 55 Preliminary data on Meledic-Mânzăleşti Natural Reserve (Buzău county, Romania) Daciana SAVA, Mariana ARCUŞ, Elena DOROFTEI……………………………………………………… 61 Contributions to the biometrical and phytobiological study on wild garlic Mariana LUPOAE, Dragomir COPREAN, Rodica DINICĂ, Paul LUPOAE………………………………. 67 Dinitrophenyl derivates action on wheat germination Cristina Amalia DUMITRAŞ -HUŢANU ………………………….……………………………………….. 73 The action of some insecticides upon physiological indices in Rana (Pelophylax) ridibunda Alina PĂUNESCU, Cristina M. PONEPAL, Octavian DRĂGHICI, Alexandru G. MARINESCU.............. 79 Changes of some physiological parameters in Prussian carp under the action of some fungicide Maria C. PONEPAL, Alina PĂUNESCU, Alexandru G. MARINESCU, Octavian DRĂGHICI................... 83 Cytogenetic effects induced by manganese and lead microelements on germination at Triticum aestivum L. Elena DOROFTEI, Maria Mihaela ANTOFIE, Daciana SAVA, Marioara TRANDAFIRESCU................... 89 Problems of the harmonizing environmental legislation at the compartment “Pisces” in the Republic of Moldova Petru COCIRTA, Olesea GLIGA ………………………………………………………………………….... 99 Biodiversity conservation in Constanţa county Silvia TURCU, Marcela POPOVICI, Loreley JIANU..................................................................................... 107 ISSN-1453-1267 © 2010 Ovidius University Press Ovidius University Annals of Natural Sciences, Biology – Ecology Series, Volume 14 (2010) The present situation of the nose horned viper populations (Vipera ammodytes montandoni Boulenger 1904) from Dobrudja (Romania and Bulgaria) Marian TUDOR……………………………………………………………………………………………… 115 Body size variation in Rana temporaria populations inhabiting extreme environments Rodica PLĂIAŞU, Raluca BĂNCILĂ, Dan COGĂLNICEANU…………………………………………… 121 Utilization of epifluorescence microscopy and digital image analysis to study some morphological and functional aspects of prokariotes Simona GHIŢĂ, Iris SARCHIZIAN, Ioan ARDELEAN……………………………………………………. 127 Changes in bacterial abundance and biomass in sandy sediment microcosm supplemented with gasoline Dan Răzvan POPOVICIU, Ioan ARDELEAN................................................................................................. 139 The formation of bacterial biofilms on the hydrophile surface of glass in laboratory static conditions: the effect of temperature and salinity Aurelia Manuela MOLDOVEANU, Ioan I. ARDELEAN............................................................................... 147 The clinical utility of additional methods in effusions evaluation Ana Maria CREŢU, Mariana AŞCHIE, Diana BADIU, Natalia ROŞOIU………………………………….. 157 Spatio-temporal dynamics of phytoplankton composition and abundance from the Romanian Black Sea coast Laura BOICENCO…………………………………………………………………………………………… 163 Aspects regarding the biodiversity of the aquatic and semi-aquatic heteroptera in the lakes situated in the middle basin of the Olt River Daniela Minodora ILIE..................................................................................................................................... 171 Program of prevention and control of fungus infestation of grain and fodder, human and animal protection against mycotoxins Ioan Aurel POP, Augustin CURTICĂPEAN, Alin GULEA, Cornel PODAR, Iustina LOBONTIU.............. 177 Data on the dynamics of some microbial groups in soils with different trophic status in Cumpăna region (Dobrudja) Elena DELCĂ………………………………………………………………………………………………... 181 The agricultural potential of phosphogypsum waste piles Lucian MATEI……………………………………………………………………………………………….. 185 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 LIMITATIVE MYCOTIC FACTORS FOR SOME PLANTS FROM THE BULGARIAN COAST OF THE BLACK SEA Gavril NEGREAN Universitatea din Bucureşti, Grădina Botanică, Şoseaua Cotroceni nr. 32, Bucureşti __________________________________________________________________________________________ Abstract: We present a list of 119 parasitic fungi collected in Bulgaria from the following groups: Peronosporales, Ascomycetes, Uredinales, Ustilaginales, Agaricales, Polyporales, Gasteromycetales and Fungi Anamorphici. An alien rust new for Bulgaria is also found (Puccinia komarovii). There are also some commentaries regarding the rare plants guested by different fungi; other fungi may contribute to the diminishing of the damages produces by some weeds; we draw attention about some foreign fungi with invasive character. Keywords: New parasitic fungi for Bulgaria, invasive fungi, matrix nova, Dobrogea, Bulgaria. __________________________________________________________________________________________ 1. Introduction. Following our preoccupa-tion regarding the limitative factors for the vascular plants on the Black Sea, we present the results of our investigations on the Bulgarian Dobrogean Black Sea side. Our observations from the previous years were published within several notes [1, 2, 3, 4, 19]. 2. Material and Methods. The fungi were collected from the areas nearby the sea side between Duranculac and the embouchure of the Batovo valley, in April and June 2006 and April – October 2008. A very small amount of fungi collectetd from other areas of Bulgaria, they also listed. The big majority are coming from the Dobrich district. The materials were collected on the way and their conditioning was donje in conformity with the usual techniques and determinated by help of the instruments we had at our disposal [5, 6, 7, 8, 9, 10, 11, 12, 13, 14]. The nomenclature of the authors of the hosts after Flora Romaniae [15] and Flora Europaea [16, 17]. The conditionated and determined materials were deposited in the Herbarium of the University from Bucureşti [BUC] and partially in the Herbarium of the Botanic Institute from Sofija [SOM]. The list is alphabetically coordinated, on ISSN-1453-1267 big groups offungi and the coronims from North to South. 3. Results and Discussions In these two years mentioned, there were collected 217 specimens, representing the analized groups of fungi (Table 1). Apparently, a number of 16 combinations fungus – host plant („matrix nova”), incase of the Peronosporales (Table 2), and were not indicated since Bulgaria [14]. Among Erysiphaceae, 19 combinations [8], alike species have not been found in Bulgaria. Likewise, a number of 15 combinations between Uredinales [7] do not seem to be cited from Bulgaria. Puccinia komarovii rust, guesting the alien plant Impatiens parviflora DC. is new for the Bulgarian mycobiota. Sozological aspects. Following a long cohabitation (co evolution) between fungi and their hosts there has been created an equilibrum, so that we have barely noticed ruptures of this equilibrum. Among the rare guested plants, we mention the following: Astragalus cornutus (important damages locally), Buglossoides arvensis subsp. sibthorpiana, Centaurea salonitana, Centaurea thracica, Clypeo-la jonthlaspi, Dianthus leptopetalus, Euphorbia myrsinites, Gypsophila pallasii, Hieracium bauhinii, Leymus racemosus subsp. sabulosus, Limonium © 2010 Ovidius University Press Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) meyeri, Onosma rigidum, Potentilla taurica, Rhagadiolus stellatus, Scilla bithynica, Sherardia arvensis L. subsp. maritima etc. Some plants are extremely important from the sozological point of view, taking into consideration that they are in contact with new climatic conditions being subjected to the eventual speciation phenomena. It will be the case of Astragalus cornutus, Beta trigyna, Carduus pycnocephalus, Euphorbia dobrogensis, Medicago arabica, Pimpinella peregrina, Plumbago europaea, Ranunculus oxyspermus, Rumex tuberosus subsp. tuberosus, Scilla bithynica etc. Some fungi can have a certain play in diminishing the population of some weeds, contributiong this way to the diminution of the damages they produce, such as Amaranthus retroflexus, Avena fatua, Bassia scoparia, Carduus acanthoides, Lycium barbarum, Malva sylvestris, Picris echioides (carantin plant), Rumex patientia etc. I collecting some alien fungi with invasiv character in present (Puccinia malvacearum, Erysiphe mougeotii, Puccinia helianthi) or in future (Puccinia komarovii, Puccinia pelargonii-zonalis). The hyperparasite Ampelomyces quisqua-lis, also contributes the diminution of the damnages produced by some mildew, there have been registered some cases. Last but not least, we consider that the fungi have their right to live. LIST OF SPECIES PERONOSPORALES Albugo amaranthi (Schwein.) O. Kuntze (Wilsonia bliti (Biv.) Thines), matrix: Amaranthus retroflexus L. - Camen Briag, centrum, in locis ruderalis, 43º27′20.83″N, 28º33′04.63″E, alt. circa 35m, 23 X 2008, G. Negrean (11.594) [BUC]. Cavarna S, prope hotel, in locis ruderalis, 43º21′17.41″N, 28º21′17.41″E, alt. circa 30 m, 10 VIII 2008, G. Negrean (11.484) [BUC]. Albugo candida (Pers.) Roussel, matrix: Alyssum desertorum Stapf - Bălgarevo E, Cap Caliacra W 2 km, in herbosis et petrosis, 14 IV 2006, G. Negrean (7065c) [BUC]. Alyssum hirsutum Bieb. - Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 11 IV 2008, G. Negrean (10.220) [BUC]. Camelina rumelica Velen. - Cavarna E, in herbosis, 12 IV 2008, G. Negrean (10.256) [BUC]. Capsella bursa-pastoris (L.) Medicus - Balcic, centrum, in cortis moscheii, ruderal, 13 IV 2006, G. Negrean (7043) [BUC]. Balcic, centrum, ruderal, 4 VI 2006, G. Negrean (7252) [BUC]. Sofija S, prope Hotel Vitosha (N), ruderal, 24 VI 2006, G. Negrean (7370). Clypeola jonthlaspi L. - Bălgarevo E, ut Cap Caliacra, in herbosis, 14 IV 2006, G. Negrean (7072a) [BUC]. Cavarna E, in herbosis, 43º24′25.14″N, 28º22′19.22″E, alt. circa 60 m, 12 IV 2008, G. Negrean (10.291) [BUC]. Sisymbrium loeselii L. - Sofija S, prope Univ. Technica, 21 VI 2006, G. Negrean (7323) [BUC]. Sofija S, prope Hotel Vitosha (N), ruderal, 24 VI 2006, G. Negrean (7382) [BUC]. Sisymbrium orientale L. s. l. - Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 9 V 2008, G. Negrean (10.436) [BUC]. Albugo portulacae (DC. ex Duby) O. Kuntze, matrix: Portulaca oleracea L. subsp. oleracea - Sozopol, in arenosis, 42º24′05.22″N, 27º42′33.32″E, alt. circa 15 m, 9 VIII 2008, G. Negrean (11.888) [BUC]. 4. Conclusions. From the 115 fungi collec-ted from the seaside of the Bulgarian Dobrogea, the most majority are plants parasites. Most of them belong the groups: Peronsporales, Erysiphaceae, Uredinales and Fungi Anamorphici. The results are important ones: a new adventitious parasite rust for the Bulgarian mycobiota and a number of 50 combinations from Bulgaria apparently not odentiofied in their form by now. We ascertained that following a long coexistence, between fungi and their hosts, the plants, there has been created a rather stable equilibrium. 4 Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) Papaver dubium L. - Bălgarevo E, Cap Caliacra, vers E, ut Mare Nigrum, in herbosis ruderalis, 43º22′03.97″N, 28º27′57.08″E, alt. circa 25 m, 15 IV 2006, G. Negrean (7079) [BUC]. Peronospora astragalina Syd., matrix: Astragalus hamosus L. - Crapetz E, prope Cap Crapetz, solo arenoso, 43º38′23.64″N, 28º34′25.86″E, alt. circa 6 m, 12 IV 2008, G. Negrean (10.242) [BUC]. Peronospora calotheca de Bary, matrix: Galium aparine L. - Crapetz E, prope Cap Crapetz, solo arenoso, 43º38′23.64″N, 28º34′25.86″E, alt. circa 6 m, 12 IV 2008, G. Negrean (10.246) [BUC]. Bălgarevo SE, Vallis Bolata, ad saxa calcarea, solo terra-rossa, 43º22′59.77″N, 28º28′20.77″E, alt. circa 15 m, 12 IV 2008, G. Negrean (10.270) [BUC]. Peronospora conglomerata Fuckel, matrix: Erodium ciconium (L.) L’Herit. - Cavarna S, in arenosis, sub Collina Cheracman, 12 IV 2008, G. Negrean (10.241) [BUC]. Cavarna SW, Bojorets S, Caliacria, in locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22 X 2008, G. Negrean (11.588) [BUC]. Peronospora farinosa (Fr.) Fr., matrix: Bassia scoparia (L.) A. J. Scott, Sofija S, cartier S, ruderal, 22 VI 2006, G. Negrean (7330) [BUC]. Chenopodium album (Boiss.) Kuntze - Camen Briag, Motel, in locis ruderalis, 43º27′13.83″N, 28º33′04.96″E, alt. circa 25 m, 7 VI 2008, G. Negrean (10.707) [BUC]. Chenopodium opulifolium Schrad. ex Koch & Ziz - Cavarna S, prope hotel, in locis ruderalis, 43º21′17.41″N, 28º21′17.41″E, alt. circa 30 m, 10 VIII 2008, G. Negrean (11.484) [BUC]. Peronospora ficariae L.R. Tul. ex de Bary, matrix: Ranunculus ficaria L. subsp. calthifolius (Reichenb.) Arcangeli - Balcic W, in locis umbrosis, 15 IV 2006, G. Negrean (7090) [BUC]. Peronospora medicaginis-minimae Gaponenko, matrix: Medicago lupulina L., Sofija S, prope Hotel Vitosha, ruderal, 22 VI 2006, G. Negrean (7321) [BUC]. Peronospora sherardiae Fuckel, matrix: Sherardia arvensis L. subsp. maritima (Griseb.) Soják - Bălgarevo E, Cap Caliacra, in herbosis, 43º22′03.97″N, 28º26′57.08″E, alt. circa 25 m, 12 Albugo tragopogonis (DC.) Gray, matrix: Xeranthemum annuum L. - Cavarna E, in herbosis, 43º24′25.14″N, 28º22′19.22″E, alt. circa 60 m, 12 IV 2008, G. Negrean (11.504) [BUC]. Ecrene N, ad oram rivuli Batova, in arenosis, 43º21′10.43″N, 28º04′31.74″E, alt. circa 2 m, 13 IV 2006, G. Negrean (7050) [BUC]. Bremia lactucae Regel, matrix: Carduus acanthoides L. - Bălgarevo E, Cap Caliacra, vers E, ut Mare Nigrum, in herbosis ruderalis, 15 IV 2006, G. Negrean (7074a) [BUC]. Cavarna SW, Bojorets S, Caliacria, in locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22 X 2008, G. Negrean (11.521) [BUC]. Picris echioides L. - Camen Briag, in locis ruderalis, 43º27′18.92″N, 28º33′14.19″E, alt. circa 35 m, 23 X 2008, G. Negrean (11.551) [BUC]. Rhagadiolus stellatus (L.) Gaertn. - Rusalca, sub platous prope littore Mare Nigrum, in abruptis et silvis, 43º24′733″N, 28º29′780″E, alt. circa 60 m, 10 V 2008, G. Negrean (10.459) [BUC]. Crepis pulchra L. - Bălgarevo E, Cap Caliacra N, sinistra vallis Bolata-Dere, in herbosis, 43º22′59.37″N, 28º28′18.08″E, alt. circa 2 m, 4 VI 2006, G. Negrean (7397) [BUC]. Hyaloperonospora parasitica (Pers.: Fr.) Constant., matrix: Alyssum desertorum Stapf - Bălgarevo E, Cap Caliacra W 2 km, in herbosis et petrosis, 14 IV 2006, G. Negrean (7065b) [BUC]. Hyaloperonospora tribulina (Pass.) Constant. (Peronospora tribulina Pass.), matrix: Tribulus terrestris L. - Cavarna SW, Caliacria, in locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22 X 2008, G. Negrean (11.511) [BUC]. Peronospora aestivalis H. Syd., matrix: Melilotus sp. - Balcic W, in herbosis, 3 VI 2006, G. Negrean (7225) [BUC]. Peronospora alsinearum Casp., matrix: Stellaria media (L.) Vill. s. l., Ecrene N, ad oram rivuli Batova, 43º21′10.43″N, 28º04′31.74″E, alt. circa 2 m, 13 IV 2006, G. Negrean (7820) [BUC]. Peronospora alta Fuckel, matrix: Plantago major L. subsp. major, Sofija S, prope Hotel Moskva, 22 VI 2006, G. Negrean (7328) [BUC]. Peronospora arborescens (Berk.) de Bary, matrix: 5 Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) Quercus pubescens Willd. - Bălgarevo E, Rusalca, prope littore Mare Nigrum, sub abruptum, 10 VIII 2008, G. Negrean (11.503) [BUC]. Quercus robur L., Sofija S, prope Hotel Vitosha (N), ruderal, 24 VI 2006, G. Negrean (7379). Erysiphe aquilegiae DC., matrix: Aquilegia vulgaris L., - Camen Briag, centrum, in locis ruderalis, subspont., 43º27′20.83″N, 28º33′04.63″E, alt. circa 35 m, 23 X 2008, G. Negrean (11.536, T) [BUC; CL]. Erysiphe artemisiae Grev., matrix: Artemisia vulgaris L. - Camen Briag, centrum, in locis ruderalis, 43º27′20.83″N, 28º33′04.63″E, alt. circa 35 m, 23 X 2008, G. Negrean (11.532) [BUC]. Erysiphe astragali DC., matrix: Astragalus hamosus L. - Rusalca, prope littore Mare Nigrum, in herbosis, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.660) [BUC]. Erysiphe buhrii U. Braun, matrix: Gypsophila pallasii Ikonn. - Bălgarevo E, dextra vallis Bolata Dere, prope littore Mare Nigrum, in herbosis, 43º23′140″N, 28º28′000″E, alt. circa 35 m, 20 VII 2006, G. Negrean (11.450) [BUC]. Erysipe cichoracearum DC., matrix: Centaurea salonitana Vis. - Rusalca N, ad littore Mare Nigrum, in herbosis, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.720) [BUC]. Yailata, platou prope littore Mare Nigrum, in saxosis, 43º26′552″N, 28º32′930″E, alt. circa 25 m, 8 VIII 2008, G. Negrean (11.472) [BUC]. Bălgarevo E, Cap Caliacra, in herbosis, 43º22′982″N, 28º26′4599″E, alt. circa 72 m, 8 VI 2008, G. Negrean (10.753) [BUC]. Balcic E, supra Tuzlata, in herbosis abruptis, 8 VI 2008, G. Negrean (10.747) [BUC]. Crepis foetida L. subsp. rhoeadifolia (Bieb.) Čelak., Sofija S, prope Hotel Vitosha (N), ruderal, 24 VI 2006, G. Negrean (7375). Crepis pulchra L., Sofija S, prope Hotel Vitosha (N), ruderal, 24 VI 2006, G. Negrean (7371). Lactuca viminea L. - Rusalca, prope littore Mare Nigrum, in locis herbosis et petrosis, 43º24′733″N, 28º29′776″E, alt. circa 45 m, 10 V 2008, G. Negrean (10.454) [BUC]. Tragopogon dubius Scop., Sofija S, prope Hotel Vitosha (N), ruderal, 24 VI 2006, G. Negrean (7376). IV 2008, G. Negrean (10.234) [BUC]. Balcic W, in herbosis, 15 IV 2006, G. Negrean (7087a) [BUC]. Peronospora tribulina Pass. = Hyaloperonospora tribulina (Pass.) Constant. Peronospora valerianellae Fuckel, matrix: Valerianella sp. - Crapetz E, prope Cap Crapetz, solo arenoso, 43º38′23.64″N, 28º34′25.86″E, alt. circa 6 m, 12 IV 2008, G. Negrean (10.245) [BUC]. Peronospora viciae (Berk.) de Bary, matrix: Vicia sativa L. subsp. nigra (L.) Ehrh. Duranculac S, in herbosis, 43º39′53.06″N, 28º31′15.26″E, alt. c. 12 m, 13 IV 2006, G. Negrean (7036) [BUC]. Rusalca NNE, in herbosis, 43º25′34.94″N, 28º31′51.46″E, alt. circa 8 m, 12 IV 2008, G. Negrean (10.277) [BUC]. Plasmopara nivea (Unger) J. Schröt. (Plasmopara umbelliferarum (Casp.) J. Schröt. ex Wartenw.), matrix: Aegopodium podagraria L., Sofija S, Montes Vitosha, in herbosis subalpinis, 23 VI 2006, G. Negrean (7353) [BUC]. ASCOMYCOTA Blumeria graminis (DC. ) Speer, matrix: Avena fatua L. - Rusalca, sub platou prope littore Mare Nigrum, in herbosis, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.646) [BUC]. Aegilops lorentii Hochst. - Rusalca, sub platou prope littore Mare Nigrum, in herbosis, 43º24′750″N, 28º29′790″E, alt. circa 15 m, 10 V 2008, G. Negrean (10.465) [BUC]. Hordeum bulbosum L. Dobrogea, Shabla E, ad littore Mare Nigrum, in locis herbosis, 43º33′707″N, 28º35′553″E, alt. circa 2 m, 9 V 2008, G. Negrean (11.572) [BUC]. Daldinia concentrica (Bolton) Ces. & De Not. - matrix: in lignos, Duranculac E, ad littore Mare Nigrum, in locis arenosis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 11 IV 2008, Leg. P. Anastasiu, det. G. Negrean (11.593) [BUC]. Epichloe typhina (Pers.: Fr.) Tul., matrix: Dactylis glomerata L. s. l., Sofija S, prope Hotel Moskva, in herbosis, 22 VI 2006, G. Negrean (7331). Erysiphe alphitoides (Griffon & Maubl.) U. Braun & S. Takam. (Microsphaera alphitoides Griffon & Maubl.), matrix: 6 Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) Erysiphe cruciferarum Opiz ex L. Junell, matrix: Alliaria petiolata (Bieb.) Cavara & Grande, Sofija S, prope Hotel Moskva, ruderal, 22 VI 2006, G. Negrean (7324). Alyssum hirsutum Bieb. - Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 11 IV 2008, G. Negrean (10.221) [BUC]. Brassica nigra C. Koch - Bălgarevo SE, ad littore Mare Nigrum, sub abruptum, prope ferma pisciculturae, in locis herbosis, 43º25′02.83″N, 28º31′00.18″E, alt. circa 5 m, 10 VIII 2008, G. Negrean (11.495) [BUC]. Erysiphe cynoglossi (Wallr.) U. Braun, matrix: Buglossoides arvensis (L.) I. M. Johnston subsp. sibthorpiana (Griseb.) R. Fernandes - Balcic W, Cap Caliacra, in herbosis, 3 VI 2006, G. Negrean (7295) [BUC]. Echium italicum L. subsp. pyramidatum (DC.) . Bălgarevo SE, Cap Caliacra, in herbosis, 43º23′02.13″N, 28º26′53.26″E, alt. circa 30 m, 10 VIII 2008, G. Negrean (11.480) [BUC]. Echium vulgare L., Sofija S, prope Hotel Vitosha (N), ruderal, 25 VI 2006, G. Negrean (7383) [BUC]. Onosma rigidum Ledeb. - Yailata, prope littore Mare Nigrum, in abruptis, 43º26′552″N, 28º32′930″E, alt. circa 45 m, 7 VI 2008, G. Negrean (11.124, A) [BUC]. Erysiphe depressa (Wallr.) Schltdl – A, matrix: Onopordum acanthium L. - Cavarna SW, Bojorets S, Caliacria, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22 X 2008, G. Negrean (11.525) [BUC]. Erysiphe galeopsidis DC. = Neoerysiphe galeopsidis (DC.) U. Braun Erysipe heraclei DC., matrix: Myrrhoides nodosa (L.) Cannon - Rusalca, sub platou prope littore Mare Nigrum, in herbosis, 43º24′750″N, 28º29′790″E, alt. circa 15 m, 10 V 2008, G. Negrean (10.465) [BUC]. Scandix pecten-veneris L. subsp. pecten-veneris Rusalca, sub platou prope littore Mare Nigrum, in herbosis, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.650) [BUC]. Tordylium maximum L. - Rusalca, sub platou prope littore Mare Nigrum, in fossa viam, 43º25′120″N, 28º31′125″E, alt. circa 12 m, 12 IV 2008, G. Negrean (11.413) [BUC]. Torilis nodosa (L.) Gaertner - Rusalca, ad littore Mare Nigrum, in herbosis prope marem, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.784) [BUC]. Erysiphe knautiae Duby, matrix: Knautia arvensis (L.) Coulter, Sofija S, prope Hotel Vitosha, in herbosis, 22 VI 2006, G. Negrean (7335) [BUC]. Erysiphe lycopsidis R. Y. Zheng & G. Q. Chen, matrix: Anchusa arvensis (L.) Bieb. - Shabla E, ad littore Mare Nigrum, in locis herbosis, 43º24′954N″, 28º30′061″E, alt. circa 10 m, 6 VI 2008, G. Negrean (10.641) [BUC]. Erysiphe mougeotii (Lév.) de Bary, matrix: Lycium barbarum L. - Cavarna, centrum, 43º26′13.44″N, 28º20′36.38″E, alt. circa 127 m, 23 X 2008, G. Negrean (11.543) [BUC]. Erysiphe polyphaga Hammarl. = Golovinomyces orontii (Castagne) V. P. Heliuta Erysiphe ranunculi Grev., matrix: Ranunculus constantinopolitanus (DC.) D’Urv. Ecrene N, ad oram rivuli Batova, 43º21′10.43″N, 28º04′31.74″E, alt. circa 2 m, 3 VI 2006, G. Negrean (7236) [BUC]. Erysiphe thesii L. Junell, matrix: Thesium alpinum L., Sofija S, Montes Vitosha, in herbosis subalpinis, 23 VI 2006, G. Negrean (7347) [BUC]. Erysiphe trifolii Grev., matrix: Medicago arabica L. - Camen Brjag, centrum, in locis ruderalis, 43º27′20.83″N, 28º33′04.63″E, alt. circa 35 m, 23 X 2008, G. Negrean (11.531, A) [BUC]. Melilotus officinalis (L.) Pallas - Cavarna SW, Bojorets S, Caliacria, in locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22 X 2008, G. Negrean (11.522) [BUC]. Sofija S, prope Hotel Moskva, in herbosis, 22 VI 2006, G. Negrean (7333) [BUC]. Trifolium hybridum L. subsp. elegans (Savi) Aschers. & Graebn., Sofija S, Hortus Botanicus, in herbosis, 20 VI 2006, G. Negrean (7313) [BUC]. Golovinomyces orontii (Castagne) V. P. Heliuta (Erysiphe polyphaga Hammarl.), matrix: 7 Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) Sedum sarmentosum Bunge - Camen Brjag, motel, cult., 43º27′20.40″N, 28º33′13.07″E, alt. circa 35 m, 7 VI 2008, G. Negrean (10.697) [BUC]. Neoerysiphe galeopsidis (DC.) U. Braun (Erysiphe galeopsidis DC.), matrix: Lamium amplexicaule L. Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300E, alt. circa 5 m, 9 V 2008, G. Negrean (10.434) [BUC]. Cavarna S, prope hotel, in locis ruderalis, 43º24′50.69″N, 28º21′16.92″E, alt. circa 20 m, 12 IV 2008, G. Negrean (10.250) [BUC]. Balcic, centrum, ruderal, 4 VI 2006, G. Negrean (7252) [BUC]. Sofija, centrum, 20 VI 2005, G. Negrean (6136) [BUC]. Phyllactinia guttata (Wallr.: Fr.) Lév., matrix: Fraxinus angustifolia Vahl subsp. oxycarpa (Bieb. ex Willd.) Franco & Rocha Afonso Cavarna, centrum, 43º26′13.44″N, 28º20′36.38″E, alt. circa 127 m, 23 X 2008, G. Negrean (11.549) [BUC]. Podosphaera euphorbiae (Castagne) U. Braun & S. Takam., matrix: Euphorbia esula L. subsp. orientalis (Boiss.) Molero & Rovira, 1992 (Euphorbia esula subsp. tommasini-ana (Bertol.) Nyman) - Bălgarevo SE, Cap Caliacra, in herbosis, 43º23′02.13″N, 28º26′53.26″E, alt. circa 70 m, 10 VIII 2008, G. Negrean (11.486) [BUC]. Sawadea bicornis (Wallr.: Fr.) Homma (Uncinula bicornis (Wallr.: Fr.) Lév., matrix: Acer negundo L., subspont., Sofija S, prope Univ. Technica, 21 VI 2006, G. Negrean (7319) [BUC]. Sphaerotheca aphanis (Wallr.) U. Braun, matrix: Geum urbanum L., Sofija S, prope Hotel Moskva, 22 VI 2006, G. Negrean (7324) [BUC]. Sphaerotheca fugax Penz. & Sacc., matrix: Erodium ciconium (L.) L’Hér. - Bălgarevo E, Cap Caliacra, in herebosis, 3 VI 2006, G. Negrean (7256) [BUC]. Geranium rotundifolium L. - Bălgarevo E, Cap Caliacra, vers E, ut Mare Nigrum, in herbosis ruderalis, 15 IV 2006, G. Negrean (7071a) [BUC]. Bălgarevo SE, Vallis Bolata, ad saxa calcarea, solo terra-rossa, 43º22′59.77″N, 28º28′20.77″E, alt. circa 15 m, 12 IV 2008, G. Negrean (10.267) [BUC]. Taphrina deformans (Berk.) Tul., matrix: Prunus dulcis Miller - Balcic, prope Hortus Botanicus, cult., 2 V 2008, G. Negrean (10.343) [BUC]. Prunus persica (L.) Batsch - Bălgarevo E, Cap Caliacra, cult., 30 IV 2008, G. Negrean (10.358) [BUC]. Taphrina pruni Tul., matrix: Prunus cerasifera Ehrh. - Shabla E, prope littore Mare Nigrum, 43º33′755″N, 28º35′250″E, alt. circa 3 m, 9 V 2008, G. Negrean (11.112) [BUC]. Prunus domestica L. - Balcic, prope hotel Eisberg, cult., 30 IV 2008, G. Negrean (10.359) [BUC]. Venturia geranii (Fr.) G. Winter, matrix: Erodium ciconium (L.) L’Herit. - Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 11 IV 2008, G. Negrean (10.224) [BUC], 9 V 2008, G. Negrean (10.430) [BUC]. UREDINALES: Aecidium euphorbiae Link, O, I, matrix: Euphorbia agraria Bieb. - Bălgarevo E, ut Cap Caliacra, in herbosis, 14 IV 2006, G. Negrean (7064a) [BUC]. Bălgarevo E, Cap Caliacra, in herbosis, 43º22′03.97″N, 28º27′57.08″E, alt. circa 25 m, 12 IV 2008, G. Negrean (10.239 [BUC]. Euphorbia myrsinites L. - Bălgarevo E, vallis Bolata Dere, terra rossa, 43º23′08.40″N, 28º27′59.49″E, alt. circa 11 m, 12 IV 2008, G. Negrean (10.268) [BUC]. Bălgarevo E, Cap Caliacra, in herbosis, 43º22′982″N, 28º26′459″E, alt. circa 72 m, 12 IV 2008, G. Negrean (10.236) [BUC]. Euphorbia nicaeensis All. s. l. - Crapetz, ut faleza, in herbosis, 12 IV 2008, G. Negrean (10.240) [BUC]. Euphorbia seguieriana Necker - Duranculac E, ad littore Mare Nigrum, in locis arenosis, 43º41′908″N, 28 º34′300″E, alt. circa 5 m, 11 IV 2008, G. Negrean (10.217) [BUC]. Melampsora euphorbiae (Ficinus & C. Schub.) Castagne, matrix: Euphorbia helioscopia L. - Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 9 V 2008, G. Negrean (10.431, ii, iii) [BUC]. Duranculac E, ad littore Mare Nigrum, in locis 8 Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 6 VI 2008, G. Negrean (10.598) [BUC]. Rusalca, sub platou prope littore Mare Nigrum, in herbosis, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.661) [BUC]. Bălgarevo E, Cap Caliacra, in herbosis, 43º22′982″N, 28º26′459″E, alt. circa 72 m, 12 IV 2008, G. Negrean (10.233, ii, iii) [BUC]. Bălgarevo E, inter Cap Caliacra et Bolata Dere, prope littore Mare Nigrum, in herbosis, 43º22′405-23′144″N, 28º27′928-965″E, alt. circa 30 m, 11 V 2008, G. Negrean (10.497) [BUC]. Cavarna, sub Montes Cheracman, 30 IV 2008, G. Negrean (10.376) [BUC]. Phragmidium mucronatum (Pers.) Schltdl, matrix: Rosa canina L. - Rusalca, prope littore Mare Nigrum, in herbosis et petrosis, 43º24′733″N, 28º29′776″E, alt. circa 55 m, 10 V 2008, G. Negrean (10.462, i) [BUC]. Phragmidium potentillae (Pers.) P. Karst., matrix: Potentilla pedata Nestler - Bălgarevo E, Cap Caliacra, in herebosis, 4 VI 2006, G. Negrean (7294) [BUC]. Potentilla taurica Willd. - Shabla E, ad littore Mare Nigrum, in locis herbosis, 43º24′954″N, 28º30′061″E, alt. circa 10 m, 6 VI 2008, G. Negrean (10.638) [BUC]. Phragmidium sanguisorbae (DC.) Schröt., matrix: Sanguisorba minor Scop. s. l., Sofija S, prope Hotel Vitosha (N), ad viam ferream, 26 VI 2006, G. Negrean (7372) [BUC]. Phragmidium violaceum (Schultz) G. Winter, matrix: Rubus candicans Weihe ex Rchb. - Bălgarevo E, Rusalca, in herbosis, supra Mare Nigrum, 43º25′02.98″N, 28º30′50.05″E, alt. circa 20 m, 19 VII 2008, G. Negrean (11.406) [BUC]. Rubus discolor Weihe & Nees - Bălgarevo SE, prope littore Mare Nigrum, sub abruptum, prope ferma pisciculturae, in locis herbosis, 43º25′02.83″N, 28º31′00.18″E, alt. circa 5 m, 10 VIII 2008, G. Negrean (11.498, ii) [BUC]. Puccinia allii (DC.) F. Rudolphi, matrix: Allium tauricum (Besser ex Rchb.) Grossh. – Bălgarevo E, Cap Caliacra, 43º22′982″N, 28º26′459″E, alt. circa 72 m, 12 IV 2008, G. Negrean (10.288) [BUC, ii]. Allium sp. - Ecrene N, ad oram rivuli Batova, in herbosis, 43º20′55.87″N, 28º04′25.15″E, alt. circa 1 m, 3 VI 2006, G. Negrean (7235) [BUC]. Puccinia asperulae-cynanchicae Wurth, matrix: Asperula tenella Heuffel ex Boiss. - NE Bulgaria: prov. Burgas: Aitos, in petrosis, 11 VI 1973, G. Negrean [BUC]. Puccinia calcitrapae DC., matrix: Carduus pycnocephalus L. - Rusalca, sub platou prope littore Mare Nigrum, in saxosis, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.698, iii) [BUC]. Centaurea thracica (Janka) Hayek - Bălgarevo E, dextra vallis Bolata Dere, in herbosis, 43º23′144″N, 28º27′965E″, alt. circa 34 m, 6 VI 2008, G. Negrean (10.785) [BUC]. Puccinia cesatii J. Schröt., matrix: Dichanthium ischemum (L.) Roberty - Bălgarevo SE, Cap Caliacra, 43º23′02.13″N, 28º26′53.26″E, alt. circa 30 m, 10 VIII 2008, G. Negrean (11.483) [BUC]. Puccinia crepidis J. Schröt., matrix: Crepis foetida L. subsp. rhoeadifolia (Bieb.) Čelak. - Bălgarevo SE, Cap Caliacra, in herbosis ruderalis, prope Archer (Boris Caragea), 43º21′38.78″N, 28º27′55.78″E, alt. circa 8 m, 11 IV 2008, G. Negrean (10.231) [BUC]. Cap Caliacra, in herbosis, 43º22′03.97″N, 28º27′57.08″E , alt. circa 30 m, 8 VI 2008, G. Negrean (10.753) [BUC]. Puccinia dobrogensis Săvul. & O. Săvul. (?= Puccinia caucasica Savelli), matrix: Iris pumila L. - Bălgarevo E, Cap Caliacra, in herbosis, 43º22′03.97″N, 28º27′57.08″E, alt. circa 25 m, 8 VI 2008, G. Negrean (11.120) [BUC]. Puccinia gladioli (Requien) Cast., I, matrix: Valerianella costata (Steven) Betcke - Bălgarevo E, Cap Caliacra, situs archaeologicus, in herbosis, 14 IV 2006, G. Negrean (7817) [BUC]. Valerianella sp. - Bălgarevo E, Cap Caliacra, in herbosis, 43º22′03.97″N, 28º27′57.08″E, alt. circa 25 m, 12 IV 2008, G. Negrean (10.255, i) [BUC]. Cavarna E, in herbosis, 13 IV 2008, G. Negrean (10.261) [BUC]. Puccinia graminis DC., matrix: Festuca drymeja Mert. & Koch - distr. Shumen: Shumen W, Platous Shumen, in silvis, 9 Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) 43º14′55.96″N, 26º53′45.52″E, alt. circa 474 m, 18 VII 2008, G. Negrean (11.603). Puccinia helianthi Schwein., matrix: Helianthus annuus L. „Florae Pleno”, cult. Camen Briag, centrum, 43º27′20.83″N, 28º33′04.63″E, alt. circa 35 m, 23 X 2008, G. Negrean (11.537) [BUC]. Puccinia hieracii Mart., matrix: Hieracium bauhinii Besser, Sofija S, prope Hotel Vitosha, 22 VI 2006, G. Negrean, matrix conf. Krahuleć (Pruhonice) (7322) [BUC]. Puccinia isiacae (Thüm.) G. Wint., matrix: Cardaria draba (L.) Desv. subsp. draba Duranculac E, ad littore Mare Nigrum, in ruderatis, 43º41′908″N, 28 º34′300″E, alt. circa 5 m, 9 V 2008, G. Negrean (10.426, i) [BUC]. Puccinia komarovii Tranzschel, matrix: Impatiens parviflora DC. - Sofija S, Hotel Vitosha (N), Parc, subspont. ad viam ferream, 24 VI 2006, G. Negrean (7380) [BUC; SOM]. Fungus adventivus novus Bulgariae. Originally from Central Asia, alien in Europe. In Romania on Impatiens parviflora DC., subspont. in Botanical Garden of Cluj-Napoca, Valea Pârîul Ţiganilor, 46°51′46″N, 23°35′20″E, alt. 347 m, 5 VII 1993, G. Negrean [BUCM 129.306]. Puccinia lapsanae Fuckel, matrix: Lapsana communis L. - Sofija S, prope Hotel Vitosha (N), ad viam ferream, 24 VI 2006, G. Negrean (7374) [BUC]. Puccinia malvacearum Bertero ex Mont. – iii, matrix: Althaea hirsuta L. - Duranculac E, ad littore Mare Nigrum, in locis herbosis, 43º40′289″N, 28º33′922″E, alt. circa 5 m, 6 VI 2008, G. Negrean (11.121) [BUC]. Bălgarevo E, Cap Caliacra, in herbosis, 43º22′405″N, 28º27′928″E, alt. circa 30 m, 8 VI 2008, G. Negrean (10.767) [BUC]. Malva sylvestris L. - Shabla E, ad littore Mare Nigrum, in locis herbosis, 43º24′954″N, 28º30′061″E, alt. circa 10 m, 9 V 2008, G. Negrean (10.444) [BUC]. Yailata, prope littore Mare Nigrum, in abruptis, 43º26′552″N, 28º32′930″E, alt. circa 25 m, 10 V 2008, G. Negrean (11.123) [BUC]. Rusalca NNE, in herbosis, 43º25′34.94″N, 28º31′51.46″E, alt. circa 8 m, 12 IV 2008, G. Negrean (10.276) [BUC], 43º24′733″N, 28º29′776″E, alt. circa 65 m, 10 V 2008, G. Negrean (10.460) [BUC], Rusalca, ad littore Mare Nigrum, in locis herbosis, 43º24′954″N, 28º30′061″E, alt. circa 10 m, 6 VI 2008, G. Negrean () [BUC]. Balcic, centrum, in cortis moscheii, ruderal, 13 IV 2006, G. Negrean (7044) [BUC]. Nesebăr, in arenosis ruderalis ad littore Mare Nigrum, 5 VI 2006, G. Negrean (7439) [BUC]. Puccinia minussensis Thüm., matrix: Lactuca tatarica (L.) C. A. Meyer - Duranculac E, ad littore Mare Nigrum, in ruderatis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 9 V 2008, G. Negrean (10.429) [BUC], 6 VI 2008, G. Negrean (10.598) [BUC]. Puccinia pachyphloea Syd. & H. Syd., matrix: Rumex tuberosus L. subsp. tuberosus - Bălgarevo E, Cap Caliacra N, Bolata-Dere, in herbosis, 4 VI 2006, G. Negrean (7396) [BUC]. Puccinia pelargonii-zonalis Doidge, matrix: Pelargonium ×hortorum auct. - Sofija S, Hortus Botanicus, in caldaria, cult. 42º43′..N, 23º19′...E, 20 VI 2006, G. Negrean (7311) [BUC; SOM]. Puccinia phragmitis (Schumach.) Körn., matrix: Rumex patientia L. s. l. - Duranculac E, ad littore Mare Nigrum, in ruderatis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 11 IV 2008, G. Negrean (10.285, i) [BUC]. Puccinia pimpinellae (F. Strauss) Link, matrix: Pimpinella peregrina L. - Yailata, prope littore Mare Nigrum, in abruptis, 43º26′552″N, 28º32′930″E, alt. circa 25 m, 10 V 2008, G. Negrean (10.465) [BUC]. Rusalca, sub platou prope littore Mare Nigrum, in herbosis, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.665) [BUC]. Puccinia procera Dietel & Holw., matrix: Leymus racemosus (Lam.) Tzvelev subsp. sabulosus (Beb.) Tzvelev - Shabla E, ad littore Mare Nigrum, in locis herbosis, 43º33′755″N, 28º35′250″E, alt. circa 3 m, 6 VI 2008, G. Negrean (10.639, ii) [BUC]. Puccinia punctata Link, matrix: Galium verum L. subsp. verum - Bălgarevo SE, prope littore Mare Nigrum, sub abruptum, prope ferma pisciculturae, in locis herbosis, 43º25′02.83″N, 28º31′00.18″E, alt. circa 5 m, 10 VIII 2008, G. Negrean (11.505) [BUC]. Puccinia recondita Dietel & Holw., matrix: 10 Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) Aegilops cylindrica Host - Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 6 VI 2008, G. Negrean (10.601) [BUC]. Balcic W, Cap Caliacra, in herbosis, 3 VI 2006, G. Negrean (7298) [BUC]. Sofija S, prope Hotel Vitosha (N), ruderal, 24 VI 2006, G. Negrean (7377). Aegilops geniculata Roth - Rusalca, platou prope littore Mare Nigrum, in locis herbosis et petrosis, 43º24′733″N, 28º29′776″E, alt. circa 60 m, 7 VI 2008, G. Negrean (10.696, iii) [BUC]. Bălgarevo E, Cap Caliacra, in herbosis, 43º22′405″N, 28º27′928″E, alt. circa 40 m, 8 VI 2008, G. Negrean (10.765, iii) [BUC]. Anchusa sp. - Bălgarevo E, Cap Caliacra, in herbosis, 43º22′982″N, 28º26′459″E, alt. circa 72 m, 12 IV 2008, G. Negrean (10.234) [BUC]. Bromus sterilis L. subsp. elegans - Sofija S, prope Hotel Vitosha, 22 VI 2006, G. Negrean (7317) [BUC]. Echium italicum L. subsp. pyramidatum (DC.) . Bălgarevo SE, Cap Caliacra, 43º23′02.13″N, 28º26′53.26″E, alt. circa 70 m, 10 VIII 2008, G. Negrean (11.482, i) [BUC]. Clematis vitalba L.- Yailata, prope littore Mare Nigrum, in abruptis, 43º26′552″N, 28º32′930″E, alt. circa 25 m, 10 V 2008, G. Negrean (10.777, i) [BUC]. Bălgarevo E, inter Cap Caliacra et Bolata Dere, prope littore Mare Nigrum, in herbosis, 43º22′405-23′144″N, 28º27′928-965″E, alt. circa 50 m, 11 V 2008, G. Negrean (10.506) [BUC]. Ecrene N, ad oram rivuli Batova, in arenosis, 43º21′10.43″N, 28º04′31.74″E, alt. circa 2 m, 3 VI 2006, G. Negrean (7238) [BUC]. Puccinia sii-falcariae J. Schröt., matrix: Falcaria vulgaris Bernh. - Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 6 VI 2008, G. Negrean (10.597) [BUC]. Shabla E, ad littore Mare Nigrum, in locis herbosis, 43º24′954″N, 28º30′061″E, alt. circa 10 m, 9 V 2008, G. Negrean (10.441) [BUC]. Cavarna E, in herbosis, 12 IV 2008, G. Negrean (10.256) [BUC]. Bălgarevo E, inter Cap Caliacra et Bolata Dere, prope littore Mare Nigrum, in herbosis, 43º22′40523′144″N, 28º27′928-965″E, alt. circa 30 m, 11 V 2008, G. Negrean (10.497) [BUC]. Bălgarevo E, Cap Caliacra N, Bolata-Dere, in herbosis, 6 VI 2006, G. Negrean (7396) [BUC]. Puccinia tanaceti DC., matrix: Artemisia absinthium L. - Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 6 VI 2008, G. Negrean (10.594) [BUC]. Cavarna SW, Caliacria, in locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 23 X 2008, G. Negrean (11.520) [BUC]. Tranzschelia pruni-spinosae (Pers.) Dietel – (ii), iii, matrix: Prunus cerasifera Ehrh. - Duranculac NE, ad confines Bulgariae, 43º44′10.05″N, 28º33′24.68″E, alt. circa 27 m, 23 X 2008, G. Negrean (11.559) [BUC; SOM]. Uromyces dianthi (Pers.: Pers.) Niessl, matrix: Dianthus leptopetalus Willd. - Bălgarevo E, sinistra vallis Bolata Dere, prope littore Mare Nigrum, in herbosis, 43º23′140″N, 28º28′000″E, alt. circa 35 m, 20 VII 2008, G. Negrean (11.476) [BUC]. Petrorhagia prolifera (L.) P. W. Ball & Heywood - Bălgarevo E, Cap Caliacra, in herbosis, 4 VI 2006, G. Negrean (7256) [BUC]. Uromyces limonii (DC.) Lév., matrix: Limonium latifolium (Sm.) Kuntze - Yailata, ad littore Mare Nigrum, 43º26′131″N, 28º32′665″E, alt. circa 12 m, 19 VII 2008, G. Negrean (11.444) [BUC]. Limonium meyeri (Boiss.) Kuntze - Rusalca, sub platou, prope littore Mare Nigrum, in saxosis, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.690) [BUC]. Rusalca, sub platou, prope littore Mare Nigrum, in saxosis, 43º25′02.83″N, 28º31′00.18″E, alt. circa 10 m, 19 VII 2008, G. Negrean (11.434) [BUC]. Uromyces lineolatus (Desm.) Schroet., matrix: Scirpus maritimus L. subsp. maritimus - Shabla NE, ad littore Mare Nigrum, in arenosis, 43º24′954″N, 28º30′061″E, alt. circa 4 m, 20 VII 2008, G. Negrean (11.578) [BUC]. Uromyce punctatus J. Schröt., matrix: Astragalus cornutus Pallas - Bălgarevo E, dextra vallis Bolata Dere, prope littore Mare Nigrum, in herbosis, 43º23′140″N, 28º28′000″E, alt. circa 35 m, 20 VII 2008, G. Negrean (11.449) [BUC]. Uromyces rumicis (Schumach.) G. Winter, matrix: Rumex patientia L. s. l. - Camen Briag, centrum, in locis ruderalis, 43º27′20.83″N, 28º33′04.63″E, 11 Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) alt. circa 35 m, 23 X 2008, G. Negrean (11.530) [BUC]. Uromyces scutellatus (Pers.: Pers.) Lév., matrix: Euphorbia agraria Bieb. - Crapetz, ut faleza, in herbosis, 12 IV 2008, G. Negrean (10.288) [BUC]. Euphorbia dobrogensis Prodan - Duranculac E, ad littore Mare Nigrum, in herbosis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 9 V 2008, G. Negrean (11.113) [BUC]. Euphorbia nicaeensis All. s.l. - Duranculac E, ad littore Mare Nigrum, in herbosis et petrosis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 6 VI 2008, G. Negrean (10.599) [BUC]. Euphorbia seguieriana Necker - Sofija S, prope Hotel Vitosha, 22 VI 2006, G. Negrean (7320). Uromyces trifolii-repentis Liro, matrix: Trifolium hybridum L. subsp. elegans (Savi) Aschers. & Graebn., Sofija S, Hortus Botanicus, in herbosis, 20 VI 2006, G. Negrean (7310) [BUC]. Cynodon dactylon (L.) Pers. - Shabla NE, ad littore Mare Nigrum, in arenosis, 43º24′954N, 28º30′061″E, alt. circa 4 m, 20 VII 2008, G. Negrean (11.584) [BUC]. Rusalca, sub platou prope littore Mare Nigrum, in Paliuretum, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 19 VII 2008, G. Negrean (11.417) [BUC]. Bălgarevo E, dextra vallis Bolata Dere, in herbosis, 43º23′144″N, 28º27′965″E, alt. circa 34 m, 6 VI 2008, G. Negrean (10.782) [BUC]. Ustilago ornithogali (J. C. Schmidt & Kunze) J. G. Kühn, matrix: Gagea pusilla (F. W. Schmidt) Schult. & Schult. fil. - Cavarna E, in herbosis, 43º24′25.14″N, 28º22′19.22″E, alt. circa 60 m, 12 IV 2008, G. Negrean (11.444) [BUC]. Ustilago vaillantii L.-R. Tul. & C. Tul., matrix: Scilla bithynica Boiss. - Ecrene N, ad oram rivuli Batova, in Alnetum, Fraxinetum pallisiae et Salicetum, in locis humidis, 43º20′55.58″N, 28º04′06.51″E, alt. circa 2 m, 13 IV 2006, G. Negrean (7056) [BUC; CL]. USTOMYCETES AGARICALES, POLYPORALES, GASTEROMYCETALES Entyloma calendulae (Oudem.) de Bary, matrix: Calendula officinalis L., Sofija S, cult., 22 VI 2006, G. Negrean (7320). Microbotryum violaceoverrucosum (Brandenb. & Schwinn) Vánky, matrix: Silene bupleuroides Sm. s. l. - Yailata, platou prope littore Mare Nigrum, in saxosis, 43º26′552″N, 28º32′930″E, alt. circa 25 m, 8 VIII 2008, G. Negrean (11.470) [BUC]. Microbotryum violaceum (Pers.: Pers.) G. Deml & Oberw. s. l., matrix: Silene latifolia Poiret subsp. alba (Miller) Greuter & Burdet - Shabla E, ad littore Mare Nigrum, in locis herbosis, 43º24′954″N, 28º30′061″E, alt. circa 10 m, 9 V 2008, G. Negrean (11.116) [BUC; CL]. Camen Brjag, centrum, in cortis, 43º27′20.83″N, 28º33′04.63″E, alt. circa 35 m, 23 X 2008, G. Negrean (11.538) [BUC; CL]. Sorosporium saponariae F. Rudolphi, matrix: Silene bupleuroides Sm. s. l. - Yailata, platou prope littore Mare Nigrum, in saxosis, 43º26′552″N, 28º32′930″E, alt. circa 25 m, 8 VIII 2008, G. Negrean (11.471) [BUC]. Ustilago cynodontis (Pass.) P. Henn., matrix: Dendrothele acerina (Pers.: Fr.) P. A. Lemke, matrix: Acer campestre L. - Rusalca, sub platou, prope littore Mare Nigrum, in silvis, 43º25′140″N, 28º31′120″E, alt. circa 15 m, 7 VI 2008, G. Negrean (10.700) [BUC]. Fomitopsis pinicola (Sw.) P. Karst., matrix: Picea abies (L.) Karsten subsp. abies - Montes Vitosha, 23 VI 2006, G. Negrean (7824) [BUC]. Hymenochaete rubiginosa (Dicks.) Lév., matrix: Quercus robur L. - Sofija, Hotel Vitosha N, Hotel Moskva W, park, 24 VI 2006, G. Negrean (7389c) [BUC]. Lepista panaeolus (Fr.) P. Karsten - ad solum, Dobrogea, Cavarna SW, Bojorets S, Caliacria, in locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 23 X 2008, G. Negrean (12.077). Polyporus melanopus (Pers.) Fr., matrix: in lignos, distr. Shumen: Shumen W, Platous Shumen, in Fagetum, 43º14′55.96″N, 26º53′45.52″E, alt. circa 474 m, 18 VII 2008, G. Negrean (11.604) [BUC]. 12 Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) Polyporus varius Pers.: Fr., matrix: ad ramulos decidous, distr. Shumen: Shumen, park, 43º16′12.73″N, 26º560′30.79″E, alt. circa 205 m, 18 VI 2008, G. Negrean (11.586) [BUC]. Suillus bellinii (Inzenga) Watling, ad solum, sub Pinus nigra Arnold, cult., Dobrogea, Cavarna SW, Bojorets S, Caliacria, in abruptis et petrosis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22 X 2008, G. Negrean (11.592) [BUC; CL]. Trametes hirsuta (Wulfen) Pilát, matrix: in lignos, distr. Shumen: Shumen W, Platous Shumen, in Fagetum, 43º14′55.96″N, 26º53′45.52″E, alt. circa 474 m, 18 VII 2008, G. Negrean (11.605) [BUC]. Tulostoma brumale Pers.: Pers., ad solum, Duranculac E, ad littore Mare Nigrum, in arenosis maritimis, 43º40′296″N, 28º33′918″E, alt. circa 5 m, 9 V 2008, comm. P. Anastasiu, det. G. Negrean & P. Anastasiu (10.426) [BUC]. Tulostoma squamosum Pers. - Ecrene N, ad oram rivuli Batova, in arenosis, 43º21′10.43″N, 28º04′31.74″E, alt. circa 2 m, 13 IV 2006, G. Negrean (7054) [BUC]. Volvariella gloiocephala (DC.: Fr.) Boekhout & Enderle - ad solum, Dobrogea, Cavarna SW, Bojorets S, Caliacria, in locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 23 X 2008, G. Negrean (12.075). alt. circa 30 m, 10 VIII 2008, G. Negrean (11.487) [BUC]. Cercospora taurica Tranzsch., matrix: Heliotropium europaeum L. - Camen Briag, centrum, in locis ruderalis, 43º27′20.83″N, 28º33′04.63″E, alt. circa 35 m, 23 X 2008, G. Negrean (11.591) [BUC; CL ]. Cavarna SW, Bojorets S, Caliacria, in locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22 X 2008, G. Negrean (11.529) [BUC]. Napicladium celtidis Cavara, matrix: Celtis australis L. - Cavarna S, prope hotel, in locis ruderalis, 43º21′17.41″N, 28º21′17.41″E, alt. circa 30 m, 10 VIII 2008, G. Negrean (11.489) [BUC]. Ovularia obliqua (Cooke) Oudem., matrix: Rumex patientia L. s. l. - Duranculac E, ad littore Mare Nigrum, locis ruderatis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 11 IV 2008, G. Negrean (10.218) [BUC]. Ramularia arvensis Sacc., matrix: Potentilla recta L., Sofija S, prope Hotel Vitosha, 20 VI 2006, G. Negrean (7309) [BUC]. Ramularia beticola Fautrey & Lambotte, matrix: Beta trigyna Waldst. & Kit. - Rusalca, sub platou prope littore Mare Nigrum, 43º24′750″N, 28º29′790″E, alt. circa 15 m, 10 V 2008, G. Negrean (10.464) [BUC]. Ramularia centaureae Lindr., matrix: Centaurea salonitana Vis. - Bălgarevo E, Cap Caliacra, in herbosis, 43º22′982″N, 28º26′459″E, alt. circa 72 m, 8 VI 2008, G. Negrean (10.754) [BUC]. Ramularia libanotidis Bubák, matrix: Seseli campestre Besser - Rusalca, sub platou, prope littore Mare Nigrum, 43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 2008, G. Negrean (10.693) [BUC]. Ramularia ranunculi-oxyspermi Lobik, matrix: Ranunculus oxyspermus Bieb. - Cavarna E, in herbosis, 43º24′25.14″N, 28º22′19.22″E, alt. circa 60 m, 12 IV 2008, G. Negrean (10.262) [BUC]. FUNGI ANAMORPHICI Ampelomyces quisqualis Ces., socio cum: Erysiphe cruciferarum Opiz ex L. Junell - matrix: Alyssum hirsutum Bieb. - Duranculac E, ad littore Mare Nigrum, in locis ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 11 IV 2008, G. Negrean (10.283) [BUC]. Socio cum: Erysiphe cynoglossi (Wallr.) U. Braun, matrix: Echium italicum L. subsp. pyramidatum (DC.) . Bălgarevo SE, Cap Caliacra, in herbosis, 43º23′02.13″N, 28º26′53.26″E, alt. circa 70 m, 10 VIII 2008, G. Negrean (11.481) [BUC]. Cercospora plumbaginea Sacc. & D. Sacc., matrix: Plumbago europaea L. - Cavarna S, prope hotel, in locis ruderalis, 43º21′17.41″N, 28º21′17.41″E, 5. References [1] NEGREAN Gavril, CONSTANTINESCU Ovidiu & DENCHEV Cvetomir M. 2004. 13 Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) Addition to the Peronosprales of Bulgaria. Mycologia Balcanica 1(1): 69-72. [2] NEGREAN Gavril & DENCHEV Cvetomir M. 2000. New record of Bulgarian parasitic fungi. Flora Mediterranea (Palermo) 10: 101-108. [3] NEGREAN Gavril & DENCHEV Cvetomir M. 2002. New record of fungi from bulgarian Dobrudzha. Pp. 21-24. In: N. RANDJELOVIĆ (ed.), Proceedings of Sixth Symposium on Flora of Southeastern Serbia and Adjacent Territories, July 4-7, 2000, Sokobanja, Yugoslavia. Vuk Karadžić, Niš. [4] NEGREAN Gavril & DENCHEV Cvetomir M. 2004. Addition to the Erysiphales of Bulgaria. Mycologia Balcanica 1(1): 63-66. [5] BRAUN Uwe. 1987. A monograph of the Erysiphales (powdery mildews). Beih. Nova Hedw. Heft 89. Berlin, Stuttgart: J. Cramer, 700 pp., 316 fig. [6] BRAUN Uwe. 1995. The powdery mildews (Erysiphales) of Europe. Jena: Gustav Fischer Verlag, i-iv, 1-337 pp., ill. 112, ISBN 3-33460994-4 (HB). [7] DENCHEV Cvetomir M. 1995. Bulgarian Uredinales. Mycotaxon 55: 405-465. [8] FAKIROVA Violeta Ilieva ● ФАКИРОВА Виолета Илиева. 1991. Fungi Bulgaricae 1 tomus ordo Erysiphales ● Гъбите в България 1 том разред Еrysiphales, red. princip. prof. Dr. I. Kovachevsky; edit. Simeon Vanev, Edit. Acad. Bulgaricae, Sofija, 154 pp. [9] MAJEWSKI T. 1977. Grzyby (Mycota), T. IX, Podstawczaki (Basidiomycetes), Rdzawniko we (Uredinales) I, Flora Polska, Warsawa - Kraków: Panstwowe Wydawnictwo Naukowe. 396 pp. [10] MAJEWSKI T. 1979. Grzyby (Mycota), T. XI, Podstawczaki (Basidiomycetes), Rdzawnikowe (Uredinales) II, Flora Polska, Warsawa Kraków: Panstwowe Wydawnictwo Naukowe. 463 pp. + Erata + 2 Pl. [11] SĂVULESCU T. 1953. Monografia Uredinalelor din Republica Populară Română ● Monographia Uredinalium Reipublicae Popularis Romanicae, vol. 1-2. Bucureşti: Edit. Academiei Române, 1166 pp. (vol. 1: 1-332 + ixxiv + liii Pl. + 21 Tab.; vol. 2: 333-1168. /B: 339-343/. [12] SĂVULESCU T. 1957. Ustilaginalele din Republica Populară Romînă ● Ustilaginales Reipublicae Popularis Romanicae, vol. 1-2. Bucureşti: Edit. Academiei Romîne, 1168 pp. /vol. I: 1-545 pp; vol. II: 546-1170 pp., index: 1141-1168/. [13] SCHOLLER M. 1996. Die Erysiphales, Pucciniales und Ustilaginales der Vorpommerschen Boddenlandschaft - Ökologisch-floristiche, florengeschichtliche und morphologisch-taxonomische Untersuchungen. Regensb. Mykol. Schriften 6: 1-325. [14] VANEV Simeon Georgiev, DIMITROVA Evtimia Georgieva & ILIEVA Elena Ivanova ● ВАНЕВ Симеон Георгиев, ДИМИТРОВА Евтимия Георгиева & ИЛИЕВА Елена Иванова. 1993. Fungi Bulgaricae 2 tomus ordo Peronosporales ● Гъбите в България 2 Tом разред Peronosporales. Red. principali Prof. Dr. Ivan KOVACHEVSKI, Editit tomum, Violeta FAKI-ROVA. Sofija: Edit. Academiae Scientiarum Bulgaricae, 195 pp. + Erata, 1 fig., 1 tab., 57 pl. ISBN 954-430-227-1 (t. 2). [15] SĂVULESCU T. (ed.). 1952-1976. Flora României ● Flora Romaniae. Bucureşti: Edit. Academiei Române. Vol. 1-13. [16] TUTIN T. G., BURGES N. A., CHATER A. O., EDMONDSON J. R., HEYWOOD V. H., MOORE D. M., VALENTINE D. H., WALTERS S. M. & WEBB D. A. (eds, assist. by J. R. AKEROYD & M. E. NEWTON; appendices ed. by R. R. MILL). 1993. Flora Europaea. 2nd ed. Vol. 1. Psilotaceae to Platanaceae. Cambridge: Cambridge University Press xlvi, 581 pp., illus. ISBN 0-521-41007-X (HB). [17] TUTIN T. G., HEYWOOD V. H., BURGES N. A., MOORE D. M., VALENTINE D. H., WALTERS S. M. & WEBB D. A. (eds). 19641980. Flora Europaea. Vols. 1-5. Cambridge: Cambridge University Press. [18] HOLMGREN Patricia K. & HOLMGREN Noel H. (ed.). 1992. Plant specialists index: Index to specialists in the systematics of plants and fungi based on data from Index Herbariorum (Herbaria), edition 8. Königstein: Koeltz Scientific Books, 1-394. [Regnum Vegetabile 120], ISBN 3-87429-331-9 (HB). [19] NEGREAN G. 1992. Violeta Ilieva Fakirova, Fungi Bulgaricae 1 tomus ordo Erysiphales, red. 14 Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) princip. prof. Dr. I. Kovachevsky; edit. Simeon Vanev, Edit. Acad. Bulgaricae, Sofija, 1991, 154 pp.etc. Stud. Cerc. Biol., Ser. Biol. Veg. 44(2): 196-197. /recenzie critică/. Aknowledgments We thanks Mrs. Professor Paulina Anastasiu for the help given in order to draw this material and to Dr. Krahulec (Pruhonice) for confirming the identification of Hieracium bauhinii. 15 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 THE MEDICINAL PLANTS OF PROVADIISKO PLATEAU Dimcho ZAHARIEV, Desislav DIMITROV University of Shumen Bishop Konstantin Preslavski, Faculty of Nature Sciences, 115 Universitetska Str., 9712, Shumen, Bulgaria, dimtchoz@yahoo.com _________________________________________________________________________________________ Abstract: Considerable taxonomical diversity of the medicinal plants of Provadiisko Plateau is established: 376 species of vascular plants from 261 genera and 86 families. Most families (77.91%) and genera (98.85%) are represented in small numbers – 1 to 4. The analysis of their life form indicates that the geophytes dominante, followed by the groups of the phanerophytes and the hemi cryptophytes. These biological types are represented mainly by perennial herbaceous plants (53.19%) and annual herbaceous plants (12.77%). The largest percentage species are of the circumboreal type (36.17%). Among the medicinal plants, there are 4 endemites and 29 relicts. 39 species with protection statute are described. The anthropophytes among the medicinal plants are 236 species (62.77%). Keywords: Provadiisko Plateau, medicinal plants, analysis of medicinal plants, protected species. ______________________________________________________________________________________ 1. Introduction In physiographic terms the Provadiisko Plateau belongs to the Danube hilly plain area, i.e. the Ludogorsko-Provadiiska subarea [1]. The Northern plateau border is the Provadiiska River; in the East it reaches to the Devnya Valley; in the South, the Provadiisko Plateau is separated from Roiaksko Plateau by Glavnica River; and finally, west of the Provaddisko Plateau is the Shumensko Plateau. The average altitude is 250 m. above sea level. The highest point is Sakartepe in the western parts of the plateau with its height of 389 m. The plateau is located in the Transcontinental climate region, district Dobrudjansko Plateau [2]. Winds are coming mostly from the North and Northeast. The average annual temperature is around 12°С. The average monthly temperatures are always positive. The temperature in January is the lowest (1.2°С) and in July – the highest (22.6°С). The minimum temperature rarely fall to 18°С, and the average maximum temperature reaches 27°С. The maximum rainfalls are in May and June and the minimum – in March and September. The annual amount of rainfalls is around 530 mm. Average humidity is around 76-77%; lowest in the summer (70%) and highest in the winter (82%) [3]. The soils, according to the FAO classification, are ISSN-1453-1267 two types. The first type is calcic chernozems located on the slopes and in the areas with low slope. The second type is calvaric fluvisols located in the Provadiiska Valley [4]. In terms of its flora, the plateau belongs to the region of Northeastern Bulgaria. The vegetation includes: forests of Carpinus betulus L. and Quercus cerris L., partly with Carpinus orientalis Mill.; mixed forests of Carpinus betulus L. and Quercus cerris L., partly with Quercus dalechampii Ten., Acer campestre L., etc.; mixed forests of Tilia tomentosa Moench., with Carpinus betulus L. or Quercus cerris L., partly also with Quercus dalechampii Ten., Acer campestre L., etc.; forest and shrubs of Carpineta orientalis; mixed forests of Quercus cerris L., Quercus pubescens Willd. and Cotinus coggygria Scop., partly with a secondary prevalence of Cotinus coggygria Scop.; mixed forests of Fraxinus ornus L. and Carpinus orientalis Mill., partly of secondary origin; shrubs with prevalence of Paliureta spinachristi, combined with xerothermal frass communities mostly replacing xerothermal forest communities of Quercus cerris L. and Quercus frainetto Ten.; shrub and grass steppe and xerothermal communities; xerothermal grass communities with a prevalence of Dichantieta ischaemi, Poaeta bulbosae, Poaeta concinnae, Chrysopogoneta grylli and Ephemereta; © 2010 Ovidius University Press The medicinal plants of the Provadiisko Plateau / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010) Community to protect natural habitats and of wild fauna and flora [11], Convention on mesoxerothermal grass vegetation with a prevalence of Poa bulbosa L., Loium perenne L., Cynodon dactylon (L.) Pers., partly also Dichantium ischaemum (L.) Roberty and rarely Chrysopogon gryllus (L.) Trin., mostly in the village com monlands; mesophytous grass communities (meadows), replacing forests of Ulmus minor Mill., Fraxinus oxicarpa Willd., Quercus robur L., Quercus pedunculiflora C. Koch.; farm areas, replacing forests of Fagus sylvatica ssp. moesiaca (K. Maly) Hyelmq.; farm areas, replacing forests of Quercus dalechampii Ten.; farm areas, replacing forests of Ulmus minor Mill., Fraxinus oxicarpa Willd., Quercus pedunculiflora C. Koch. [5]. The first studies of the flora of the plateau have been conducted in the 1990s by Vasil Kovachev around Madara, Kaspitchan and Provadia [6]. The results are found in the first volume on Bulgarian flora [7] and its supplement [8]. Hermengild Shkorpil also conducted botanical researchin the vicinity of Provadia in the early twentieth century [6]. So far, data on the medicinal plants in the area of Provadiisko Plateau have been published by authors for the the territory of Municipality Provadia [9] and by Zahariev and Uzunov for the protected area Madarski rock wreaths [10]. The Provadiisko Plateau is a part of the protected zone Provadiisko-Roiaksko Plateau by Natura 2000, according to Council Directive 92/43/EEC of the European Community to protect natural habitats and of wild fauna and flora [11]. International Trade in Endangered Species of Wild Fauna and Flora (CITES) [21], Red book of PR Bulgaria [22], IUCN Red List for Bulgaria [23], Biological Diversity Act [24], Order for special arrangements for the conservation and use of medicinal plants [25]. The anthropophytes are presented by Stefanov and Kitanov [26]. 3. Results and Discussions As a result of the research of the medicinal plants of the Provadiisko Plateau 376 species of vascular plants from 261 genera and 86 families have been indetified. They represent 9.83% from all species, 29.36% from all genera and 50.89% from all plant families in Bulgaria. Most families (77.91%) and genera (98.85%) are represented in small numbers: 1 to 4. Almost all families (86.05%) are represented with 1-4 genera. Only 13.95% from the families included 5 or more genera (Table 1). Most genera are found in the families: Asteraceae (28), Lamiaceae (22), Fabaceae (21), Rosaceae (15), Apiaceae (14) and Brassicaceae (12). Table 1. Families with greatest number of genera Families Asteraceae Lamiaceae Fabaceae Rosaceae Apiaceae Brassicaceae Scrophulariaceae Ranunculaceae Caryophyllaceae Boraginaceae Poaceae Solanaceae 2. Material and Methods The field studies were conducted on the route method in 2007-2009. The names of the taxons are taken from the Flora of PR Bulgaria, Vol. І – Х [12]. The update of the taxons is consistent with APG II [13]. The life forms are presented by Raunkier [14]. In their determination was used Flora of PR Bulgaria, Vol. І – Х [12]. The biological types are presented by Kozuharov [15]. The floristic elements and endemites are presented by Asiov et all. [16]. The relicts are presented by Gruev and Kuzmanov [17], Peev [18], Boža et all. [19], Peev et all. [20]. The protection status is presented using the following documents: Council Directive 92/43/EEC of the European Genera 28 22 21 15 14 12 8 8 8 7 5 5 Most families – 77.91% have 1-4 species. Only 22.09% of the families are represented by 5 or more species (Table 2). Most species belong to the following families: Asteraceae (42), Lamiaceae (41), 18 Dimcho Zahariev, Desislav Dimitrov / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010) Fabaceae (28), Rosaceae (26), Brassicaceae (15), Apiaceae (15), Ranunculaceae (12) and Scrophulariaceae (11). The mezophanerophytes are 35 species, of which essential are: Acer campestre L., Acer pseudoplatanus L., Carpinus betulus L., Fagus Almost all genera (98.85%) are represented by 14 species. Most species – more than 5 have only 1.15% of the genera (Table 3): Centaurea, Geranium and Thymus. sylvatica L., Fraxinus ornus L., Tilia tomentosa Moench, Ulmus minor Mill. The microphanerophytes are 27 species, the most common of which are: Acer tataricum L., Cornus mas L., Corylus avellana L., Cotinus coggygria Scop., Crataegus monogyna Jacq., Hedera helix L., Ligustrum vulgare L., Paliurus spina-christi Mill., Prunus spinosa L., Rosa canina L., Rubus caesius L., Sambucus nigra L. The nanophanerophytes are 11 species, which are essential: Clematis vitalba L., Genista tinctoria L., Teucrium chamaedrys L., Teucrium polium L. The succulents are represented by 3 species: Sedum acre L., Sedum album L. and Sedum maximum (L.) Suter. Table 2. Families with greatest number of species Families Asteraceae Lamiaceae Fabaceae Rosaceae Brassicaceae Apiaceae Ranunculaceae Scrophulariaceae Boraginaceae Caryophyllaceae Orchidaceae Geraniaceae Polygonaceae Solanaceae Aspleniaceae Oleaceae Poaceae Rubiaceae Salicaceae Species 42 41 28 26 15 15 12 11 9 9 7 6 6 6 5 5 5 5 5 Table 4. Life forms Group Subgroup Megaphanerophytes Mezophanerophytes Phanerophytes Microphanerophytes (Ph) Nanophanerophytes Epiphytes Succulents Hamephytes (Ch) Hemi cryptophytes (H) Therophyte – hemi cryptophytes (Th-H) Cryptophytes Geophytes (Cr) Helophytes Hydrophytes Therophytes (Th) Table 3. Genera with greatest number of species Families Asteraceae Geraniaceae Lamiaceae Genera Centaurea Geranium Thymus Species 5 5 5 Species 10 35 27 11 – 3 5 65 45 126 1 – 48 The group of hamephytes (Ch) includes 5 species: Dictamnus albus L., Ruscus aculeatus L., Satureia montana L., Thymus jankae Čelak., Thymus zygioides Griseb. The hemi cryptophytes (H) are 65 species, of which most common are: Agrimonia eupatoria L., Carlina vulgaris L., Cichorium intybus L., Clinopodium vulgare L., Echium vulgare L., Eryngium campestre L., Lotus corniculatus L., Marrubium peregrinum L., Plantago lanceolata L., In the analysis of the life forms were obtained the following results (Table 4): The phanerophytes (Ph) are represented by 86 species. The megaphanerophytes are represented by 10 species, the most common of which are: Acer pseudoplatanus L., Fraxinus excelsior L., Gleditsia triacanthos L., Pinus sylvestris L., Quercus frainetto Ten., Quercus robur L. 19 The medicinal plants of the Provadiisko Plateau / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010) Plantago media L., Polygala major Jacq., Ranunculus ficaria L., Salvia nemorosa L., Silene vulgaris (Moench) Garcke, Taraxacum officinale species (3.19%) and species from transition group from biennial to perennial plants (b-p) – 11 species (2.92%). The largest group includes annual as well as perennial plants (a-p) and is represented by 5 species (1.33%). Web., Trifolium pratense L., Trifolium repens L., Verbena officinalis L., Viola odorata L. The transition group therophytes – hemi cryptophytes (Th-H) comprises 45 species, of which essential are: Alliaria petiolata (Bieb.) Cavara et Grande, Arctium lappa L., Capsella bursa-pastoris Moench., Daucus carota L., Erodium cicutarium (L.) L̀ Her., Heracleum sibiricum L., Malva sylvestris L., Plantago major L., Stellaria media (L.) Vill., Tordylium maximum L., Verbascum densiflorum Bertol., Viola tricolor L. The group of cryptophytes (Cr) is the largest and includes 127 species. Their significant proportion can be explained by the dominance of forest habitats within the plateau. Geophytes dominate with total of 126 species; the most widespread of them are: Achillea millefolium L., Anemone ranunculoides L., Artemisia absinthium L., Artemisia vulgaris L., Chelidonium majus L., Convolvulus arvensis L., Coronilla varia L., Fragaria vesca L., Galanthus elwesii Hook. fil., Galanthus nivalis L., Geum urbanum L., Isopyrum thalictroides L., Potentilla argentea L., Sanguisorba minor Scop., Scilla bifolia L., Urtica dioica L. The helophytes is represented by one species only: Typha latifolia L. The therophytes (Th) are 48 species. The most widespread are: Galium aparine L., Lactuca serriola L., Lamium purpureum L., Lolium temulentum L., Melilotus officinalis (L.) Pall., Papaver rhoeas L., Parietaria lusitanica L., Xeranthemum annuum L. The largest group species in terms of biological types (Figure 1) are perennial plants (p) – 200 species (53.19%). Their dominance can be explained with the wide variety of communities and habitats within the plateau. The annual plants (a) are 48 species (12.77%), which can be explained by the presence of dry rocky terrain and arable lands on the plateau. The tree species (t) are 39 (10.37%). The next group includes shrubs (sh) – 29 species (7.71%). The transition group from annual to biennial plants (a-b) includes 19 species (5.05%). The biennial plants (b) are 13 species (3.46%). There are species from transition group from tree to shrubs (sh-t) with 12 39 48 12 a 19 29 a-b 5 a-p 13 b 11 b-p p sh sh-t t 200 Fig. 1. Biological types The specific physiographic conditions on the Provadiisko Plateau determined considerable diversity of floristic elements. 7 different types of floristic elements are established (Table 5). The dominant elements are elements from circumboreal type – 136 species (36.17%), followed by European elements – 101 species (26.86%) and Mediterranean elements – 67 species (17.82%). The endemic component is represented by 4 species (1.06%). It includes 3 Balkan endemites – Achillea clypeolata Sibth. et Sm., Aesculus hippocastanum L., Inula aschersoniana Janka and 1 Balkan subendemite – Syringa vulgaris L. Table 5. Floristic elements Floristic elements Circumboreal type European type Mediterranean type Pontic type Adventive type Cosmopolitan type Balkan endemic and subendemic type Other Species 136 101 67 27 20 19 4 2 This distribution can be explained by the location of the plateau in the transcontinental climate region. The proximity of the plateau to the border of a 20 Dimcho Zahariev, Desislav Dimitrov / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010) temperate region is the reason for the prevalence of circumboreal and European floristic elements. At the same time, the impact of the continentalmediterranean region in terms of the Black Sea and the karst topography create conditions for the category „Rare”: Artemisia pontica L., Cercis siliquastrum L., Juniperus sabina L., Tilia rubra DC. In the Biological Diversity Act 8 species are included in the category „Protected”: Aesculus hippocastanum L., Anacamptis pyramidalis C. Rich., Anemone sylvestris L., Galanthus elwesii Hook. fil., Galanthus development of a large number of mediterranean species. The flora of the plateau includes significant number of relict species: 29. They account for 7.71% of the total number of species. The majority of the relict species are Tertiary relicts. They are 28 species: Abies alba Mil., Acer campestre L., Acer pseudoplatanus L., Acer tataricum L., Aesculus hippocastanum L., Betula pendula Roth, Carpinus betulus L., Carpinus orientalis Mill., Celtis australis L., Cercis siliquastrum L., Clematis vitalba L., Corylus avellana L., Cotinus coggygria Scop., Fraxinus excelsior L., Fraxinus ornus L., Hedera helix L., Juniperus communis L., Picea abies (L.) Karsten, Pinus nigra Arn., Populus alba L., Populus nigra L., Ruscus aculeatus L., Salix alba L., Salix caprea L., Smilax excelsa L., Taxus baccata L., Ulmus minor Mill., Viscum album L. One of the relict species is quaternary – Galanthus nivalis L. 39 species with protection statute are described. One of them – Himantoglossum caprinum (Bieb.) C. Koch., is included in the list of species, protected by the Berne Convention and Natura 2000. In CITES 10 species are included: Adonis vernalis L., Anacamptis pyramidalis C. Rich., Galanthus elwesii Hook. fil., Galanthus nivalis L., Himantoglossum caprinum (Bieb.) C. Koch., Orchis morio L., Orchis purpurea Huds., Orchis simia L., Orchis tridentata Scop., Platanthera chlorantha (Cust.) Rchb. In the IUCN Red List for Bulgaria 5 species are included under the category „Threatened”: Aesculus hippocastanum L., Galanthus elwesii Hook. fil., Galanthus nivalis L., Juniperus sabina L., Taxus baccata L., 2 species are included under the category „Vulnerable”: Anacamptis pyramidalis C. Rich., Himantoglossum caprinum (Bieb.) C. Koch, 2 species are in the category „Nearly threatened”: Anemone sylvestris L., Cercis siliquastrum L. and 1 species is included in the category „Of least concern”: Tilia rubra DC. In the Red book for Bulgaria 4 species are included in the category „Endangered”: Aesculus hippocastanum L., Anemone sylvestris L., Galanthus nivalis L., Taxus baccata L. and 4 species are included in the nivalis L., Himantoglossum caprinum (Bieb.) C. Koch., Juniperus sabina L., Taxus baccata L. In the category “Under the protection and regulated use of nature” are 14 species: Asparagus officinalis L., Crocus pallasii Bieb., Echinops sphaerocephalos L., Gypsophila paniculata L., Helichrysum areanrium (L.) Moench., Lilium martagon L., Orchis morio L., Orchis purpurea Huds., Orchis simia L., Orchis tridentata Scop., Polygonatum odoratum (Mill.) Druce, Ruscus aculeatus L., Salix caprea L., Scilla bifolia L. Collecting herbs is prohibited from the natural habitats of 15 species: Adonis vernalis L., Althaea officinalis L., Artemisia santonicum L., Asarum europaeum L., Asplenium trichomanes L., Convallaria majalis L., Glaucium flavum Crantz, Helichrysum areanrium (L.) Moench., Orchis morio L., Orchis purpurea Huds., Orchis simia L., Orchis tridentata Scop., Phyllitis scolopendrium (L.) Newm., Ruscus aculeatus L., Valeriana officinalis L. Under a restrictive regime are 4 species: Berberis vulgaris L., Carlina acanthifolia All., Galium odoratum (L.) Scop., Sedum acre L. The anthropophytes among the medicinal plants are 236 species (62.77%). Many of them are considered weed or ruderal plants. The most common as weed are: Anagallis arvensis L., Brassica nigra (L.) Koch, Centaurea cyanus L., Chenopodium album L., Chenopodium polyspermum L., Consolida hispanica (Costa) Greut. et Burdet, Consolida regalis S. F. Gray, Cynodon dactylon (L.) Pers., Datura stramonium L., Myosotis arvensis (L.) Hill, Nigella arvensis L., Papaver rhoeas L., Senecio vulgaris L., Stellaria media (L.) Vill., Thlaspi arvense L., Xanthium strumarium L. Оf the ruderal plants most common are: Capsella bursa-pastoris Moench., Cardaria draba (L.) Desv., Chamomilla recutita (L.) Rausch., Chelidonium majus L., Conium maculatum L., Conyza canadensis (L.) Cronq., Heracleum sibiricum L., Lactuca serriola L., Parietaria lusitanica L., Sambucus ebulus L., Solanum dulcamara L., Urtica dioica L. 21 The medicinal plants of the Provadiisko Plateau / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010) [3] KOPRALEV, I. (main ed.), 2002. Geography of Bulgaria. Physical and socio-economic geography, Institute of Geography, BAS, Farkom, Sofia, 760 pp. [4] NINOV, N., 2002. Soils, in Kopralev, I. (main ed.). Geography of Bulgaria. Physical and socioeconomic geography, Institute of Geography, BAS, Farkom, Sofia, 760 pp. 4. Conclusions Considerable taxonomical diversity of the medicinal plants of Provadiisko Plateau is identified: 376 species of vascular plants from 261 genera and 86 families. Most families (77.91%) and genera (98.85%) are represented in small numbers: 1 to 4. The analysis of life forms indicates the predominnce of geophytes, followed by the groups phanerophytes and hemi cryptophytes. The biological types are represented mainly by perennial herbaceous plants (53.19%) and annual herbaceous plants (12.77%). The identified medicinal plants can be categorized into 7 types of floristic elements. The highest percentage species are of the circumboreal type (36.17%). Among the medicinal plants of Provadiisko Plateau 4 endemites and 29 relicts are described. 39 species with protection status are described: the use of 1 species is restricted by the Berne Convention and Natura 2000; 10 species are included in CITES; 10 species are included in IUCN Red List for Bulgaria; 8 species appear in the Red book for Bulgaria; 22 species are included in the Biological Diversity Act; 14 species are included in the category “Under the protection and regulated use of nature”, the collecting of herbs from their natural habitats is prohibited for 15 species, and 4 species are under a restrictive regime. The anthropophytes among the medicinal plants are 236 species (62.77%). Many of them are considered weed or ruderal plants. [5] BONDEV, I., 1991. The vegetation of Bulgaria. Map in М 1:600 000 with explanatory text, University Press St. Kliment Ohridski, Sofia, 183 pp. [6] STANEV, S., 2001. Little known names from Bulgarian botany, Pensoft, Sofia – Moscow, 202 pp. [7] VELENOVSKY, J., 1891. Flora Bulgarica, Praga, 676 рp. [8] VELENOVSKY, J., 1898. Flora Bulgarica, Supplementum I, Praga, 420 рp. [9] ZAHARIEV, D., Dimitrov D., 2009. The medicinal plants in area of Provadiisko Plato (Municipality Provadia), 8th National conference with international participation „Natural sciences – 2009”, 2-3.10.2009, Varna (upcoming). [10] ZAHARIEV, D., Uzunov G., 2009. A study of the flora in Protected place Madarski skalni venci, 8th National conference with international participation „Natural sciences – 2009”, 23.10.2009, Varna (upcoming). [11] Council Directive 92/43/EEC of the European Community to protect natural habitats and of wild fauna and flora. [12] Flora of PR Bulgaria, Vol. І-Х, 1963-1995, Publishing House of BAS, Sofia. [13] CHASE, M. (corresponding author), 2003. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II, The Linnean Society of London, Botanical Journal of the Linnean Society, 141: 399–436. [14] PAVLOV, D., 2006. Phytocoenology, Publishing House of University of Forestry, Sofia, 251 pp. [15] KOZUHAROV, S. (ed.), 1992. Identifier of the vascular plants in Bulgatia, Nauka i izkustvo, Sofia, 788 pp. 5. References [1] GALABOV, J., 1966. Main lines of the relief (Common morphographic and morphometric characteristics), in Geography of Bulgaria, Physical geography – Relief, Vol. 1, Sofia. [2] VELEV, S., 2002. Climatic zoning, in Kopralev, I. (main ed.). Geography of Bulgaria. Physical and socio-economic geography, Institute of Geography, BAS, Farkom, Sofia, 760 pp. 22 Dimcho Zahariev, Desislav Dimitrov / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010) [16] ASIOV B., Petrova A., Dimitrov D., Vasilev R., 2006. Conspectus of the Bulgarian vascular flora. Distribution maps and floristic elements, Bulgarian Biodiversity Foundation, Sofia, 452 pp. [17] GRUEV, B., Kuzmanov B., 1994 – General biogeography, University Press St. Kliment Ohridski, Sofia, 498 pp. [18] PEEV, D., 2001. National park Rila. Management plan 2001 – 2010. Adopted by Resolution №522 of Council of Ministers on 04.07.2001, Sofia, 338 pp. [19] BOŽA, P., Anačkov G., Igić R., Vukov D., Polić D., 2005. Flora “Rimskog šanca” (Vojvodina, Srbija), 8th Symposium on the flora of Southeastern Serbia and Neighbouring Regions, Niš, 20-24.06.2005, Abstracts, рр. 55. [20] PEEV, D., Kozuharov S., Anchev M., Petrova A., Ivanova D., Tzoneva S., 1998. Biodiversity of Vascular Plants in Bulgaria, In: Curt Meine (ed.), Bulgaria's Biological Diversity: Conservation Status and Needs Assessment, Volumes I and II, Washington, D.C., Biodiversity Support Program, pp. 55–88. [21] Convention on International Trade in Endangered Species of Wild Fauna and Flora, State Gazette number 6 from 21 Januari 1992. [22] Red book of PR Bulgaria, Vol. 1, Plants, 1984, Publishing House of BAS, Sofia, 447 pp. [23] PETROVA А., Vladimirov V. (eds.), 2009. Red List of Bulgarian vascular plants, Phytologia Balcanica 15 (1): 63–94. [24] Biological Diversity Act, State Gazette number 77 from 9 august 2002, pp. 9–42. Amended in State Gazette number 94 from 16 November 2007. [25] Order number RD-72 from 3 februari 2006 for special arrangements for the conservation and use of medicinal plants, State Gazette number 16 from 21 Februari 2006. [26] STEFANOV, B., Kitanov B., 1962. Kultigenen plants and kultigenen vegetation in Bulgaria, Publishing House of BAS, Sofia, 275 pp. 23 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 THE PLANTS WITH PROTECTION STATUTE, ENDEMITES AND RELICTS OF THE SHUMENSKO PLATEAU Dimcho ZAHARIEV, Elka RADOSLAVOVA University of Shumen Bishop Konstantin Preslavski, Faculty of Nature Sciences, 115 Universitetska Str., 9712, Shumen, Bulgaria dimtchoz@yahoo.com __________________________________________________________________________________________ Abstract: As a result of our investigations of the Shoumen Plateau in the period 1998-2009, 786 species were identified, of which the number of species with conservation status is 80 (10.18%). 2 of those species are included in Appendix II of Directive 92/43/ЕЕС. 24 of the species are included in CITES. 32 species are included in the IUCN Red List for Bulgaria under the following categories: threatened – 13, vulnerable – 9, nearly threatened – 5 and least concern – 5 species. In the Red book for Bulgaria, there are 7 endangered species and 14 are rare plants. In the Biological Diversity Act, 23 species are included in Appendix 3 and further 28 species – in Appendix 4. The collecting of herbs from their natural habitats is prohibited for 12 species, and 6 species are under a restriction. 29 species (3.69%) are endemites. These are 17 Balkan subendemites, 9 Balkan endemites and 3 Bulgarian endemites. The flora of the plateau includes a significant number of relict species – 42. (5.34%). The majority of them, 39 species, are Tertiary relicts, 2 are quaternary relicts and 1 is a postglacial steppe relict. Keywords: Shumensko Plateau, plants with protection statute, endemites, relicts. __________________________________________________________________________________________ 1. Introduction Shumensko Plateau refers to an area in the hills east of the Danube plain, which was declared protected by Natura 2000. This was determined by the hills’ role in support of the biodiversity among large territories of scattered forests. The majority of the Shumensko Plateau area – 3929.9 ha (53%), was declared for National Park in 1980. In 2003, the park was recognized as Nature Park. The regime of use and management of the park is determined by the Protected Areas Act [1] and the Management Plan for the Nature Park [2]. In the park is located the Bukaka Preserve. This is a forest area of 63.04 ha, declared protected due to the indigenous forest that has existed there for several centuries and is comprised of Fagus sylvatica subsp. moesiaca. On the territory of the preserve, all human activity is prohibited, except for people passing on specifically marked paths. Shumensko Plateau has been declared protected by Natura 2000 and its estimated area is 4490.62 hа. This territory is also protected under the Council ISSN-1453-1267 Directive 92/43/EEC of the European Community for protecting natural habitats of wild fauna and flora [3]. The unique combination of conditions in terms of topography, water resources, climate and soil, determine the diversity of the plant species in the area. In the past, Velenovsky and his collaborators Hermengild Shkorpil and Anani Iavashev began the study of the plateau’s flora. In the 1980s, they collected the first botanical data in Northeast Bulgaria, including the area of the Shumen vicinity [4]. Their research is presented in the first volume on the Bulgarian flora [5] and its supplement [6]. Davidov [7] conducted his own research on the flora of Shumen and the territory around the town. Further information about individual species, distributed on the plateau, can be found in Stoyanov and Stefanov [8, 9, 10], Stoyanov, Stefanov and Kitanov [11] and in Flora of PR Bulgaria, Vol. І – Х [12]. The diversity of species of the Orchidaceae family has been studied by Radoslavova [13]. In the Management Plan for the National Park Shumensko Plateau [2]: there are 550 species of vascular plants © 2010 Ovidius University Press The plants with protection statute, endemites and relicts.../ Ovidius University Annals of Biology-Ecology 14: 25-31 (2010) (i.e. mosses not counted) described in that source. Our studies [14] show that the number of vascular plants on the territory of the entire plateau is 786 species. According to the forest development project of the Shumen Forestry [15], a total of 16 species have conservation statute and are included in the Red book of PR Bulgaria [16]. Seven of the species are endangered: Aesculus hippocastanum L., Anacamptis pyramidalis C. Rich., Anemone sylvestris L., Castanea sativa Mill., Galanthus nivalis L., Himantoglossum hircinum (L.) Spreng., Paeonia tenuifolia L. Nine of the species are rare: Atropa belladonna L., Celtis caucasica Willd., Cercis siliquastrum L., Cyclamen coum Mill., Fibigia clypeata (L.) Medic., Fritillaria pontica Wahl., Haplophyllum thesioides G. Don., Jurinea ledeborii Bunge., Pastinaca umbrosa Stev. ex DC. Six of these species are protected by the Biological Diversity Act (BDA) [17]: Aesculus hippocastanum L., Anacamptis pyramidalis C. Rich., Anemone sylvestris L., Cyclamen coum Mill., Galanthus nivalis L., Himantoglossum hircinum (L.) Spreng. Six of the plateau species are listed in the Red Book of the district Shumen [18]. Two of them are endangered: Lilium martagon L. and Campanula euxina (Vel.) Ancev. Four of the species are rare: Himantoglossum hircinum (L.) Spreng., Anacamptis piramidalis (L.) Rich., Ruscus hyppoglosum L. and Galium paschale Forsskal. In the Management Plan of the National Park Shumensko Plateau [2] are found 18 species with conservation statute that are also listed in the Red book of PR Bulgaria. Five of them are in the category “endangered”: Anemone sylvestris L., Colchicum davidovii Stefanov, Galanthus nivalis L., Ruta graveolens L., Veronica spicata L. Thirteen species fall into the category “rare”: Anthemis regis-borisii Stoj. et Acht., Anthemis rumelica (Velen.) Stoj. et Acht., Celtis caucasica Willd., Cyclamen coum Mill., Erodium hoefftianum C. A. Meyer, Fibigia clypeata (L.) Medic., Fritillaria graeca Boiss. & Spruner, Fritillaria pontica Wahl., Galium bulgaricum Vel., Haplophyllum thesioides G. Don., Hedysarum tauricum Pallas ex Willd., Jurinea ledeborii Bunge., Pastinaca umbrosa Stev. ex DC. 2. Material and Methods 26 Our study of the flora of the Shoumen Plateau was conducted on the route method in 1998 – 2009. The names of the taxons are taken from the Flora of PR Bulgaria, Vol. І – Х [12]. The update of the taxons is consistent with APG II [19]. The endemites are represented by Asiov et all. [20]. The relicts are represented by Gruev and Kuzmanov [21], Peev [22], Boža et all. [23], Peev et all. [24]. The conservation statute is recognized using the following documents: Council Directive 92/43/EEC of the European Community to protect natural habitats and of wild fauna and flora [3], Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) [25], Red book of PR Bulgaria [16], IUCN Red List for Bulgaria [26], Biological Diversity Act [17], Order for special arrangements for the conservation and use of medicinal plants [27]. 3. Results and Discussion The analysis of the received data leads to the following results and conclusions: Two species, Anacamptis pyramidalis C. Rich. and Himantoglossum hircinum (L.) Spreng., of the 16 protected and listed as endangered species in the forest development project of the Shumen Forestry do not fall into any category protected by the Red Book of PR Bulgaria. They are listed as “rare” in the Red Book of the district Shumen. Furthermore, in the Red List of the Bulgarian vascular plants, they are given similar status – “ vulnerable”. Three of the species: Atropa belladonna L., Castanea sativa Mill. and Paeonia tenuifolia L. we did not find on the territory of the plateau. Himantoglossum hircinum (L.) Spreng. is incorrectly recorded as located in Bulgaria and should be replaced with the correct species name, Himantoglossum caprinum (Bieb.) C. Koch. The name Celtis caucasica Willd. is obsolete, now replaced by Celtis glabrata Steven. As a result of several years of observations, we found that populations of the following species have increased: Anacamptis pyramidalis C. Rich., Cyclamen coum Mill., Galanthus nivalis L., Himantoglossum caprinum (Bieb.) C. Koch., Lilium martagon L. and Ruta graveolens L. Therefore, they are not really endangered anymore. Dimcho Zahariev, Elka Radoslavova / Ovidius University Annals of Biology-Ecology 14: 25-31 (2010) From the 18 protected species included in the Management Plan of National Park Shumensko Plateau, 2 species, Colchicum davidovii Stefanov and Veronica spicata L., listed as endangered, have not been confirmed by us as existing on the plateau. We think that Colchicum davidovii Stefanov has disappeared from the flora of the plateau. Four species listed as rare or “rare” species, we did not found on the plateau: Anthemis rumelica (Velen.) Stoj. et Acht., Fritillaria graeca Boiss. & Spruner, Galium bulgaricum Vel., Hedysarum tauricum Pallas ex Willd. The new data for the conservation statute of the species, established by us within the realm of the Shumensko Plateau, shows the following: The total number of species with conservation statute is 80 (Figure 1). This is a 10.18% from the total number of species found on the Shumensko Plateau. We found the following species: 1. Aegilops geniculata Roth 2. Aesculus hippocastanum L. 3. Althaea officinalis L. 4. Anacamptis pyramidalis C. Rich. 5. Anemone sylvestris L. 6. Anthemis regis-borisii Stoj. et Acht. 7. Artemisia pedemontana Balb. 8. Asarum europaeum L. 9. Asparagus tenuifolius Lam. 10. Asparagus verticillatus L. 11. Asplenium trichomanes L. 12. Berberis vulgaris L. 13. Betonica officinalis L. 14. Bupleurum affine Sadl. 15. Bupleurum apiculatum Friv. 16. Bupleurum praealtum L. 17. Bupleurum rotundifolium L. 18. Campanula euxina (Vel.) Ancev 19. Carlina acanthifolia All. 20. Celtis glabrata Steven 21. Centaurea marshalliana Spreng. 22. Cephalanthera damasonium (Mill.) Druce 23. Cephalanthera longifolia (L.) Fritsch 24. Cephalanthera rubra (L.) Rich. 25. Cercis siliquastrum L. 26. Convallaria majalis L. 27. Crocus flavus West. 28. Crocus pallasii Bieb. 29. Cyclamen coum Mill. 30. Dactylorhiza saccifera (Brongn.) Soo 27 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. Dryopteris filix-mas (L.) Schott Echinops sphaerocephalos L. Epipactis helleborine (L.) Crantz Epipactis microphylla (Ehrh.) Sw. Epipactis purpurata Smith Erodium hoefftianum C. A. Mey. Fibigia clypeata (L.) Medic. Fritillaria pontica Wahl. Galanthus elwesii Hook. fil. Galanthus nivalis L. Galium odoratum (L.) Scop. Galium rubioides L. Gypsophila paniculata L. Haplophyllum thesioides G. Don. Helichrysum arenarium (L.) Mornh. Himantoglossum caprinum (Bieb.) C. Koch Juniperus sabina L. Jurinea ledebourii Bunge Lilium martagon L. Limodorum abortivum (L.) Sw. Listera ovata (L.) R. Br. Neottia nidus-avis (L.) Rich. Ophrys apifera Huds. Ophrys cornuta Stev. Ophrys mammosa Desf. Orchis morio L. Orchis purpurea Huds. Orchis simia Lam. Orchis tridentata Scop. Pastinaca umbrosa Stev. et DC. Phyllitis scolopendrium (L.) Newm. Platanthera chlorantha (Cust.) Rchb. Polygonatum odoratum (Mill.) Druce Polystichum aculeatum (L.) Roth Primula veris L. Pulmonaria mollis Horn. Ruscus aculeatus L. Ruscus hypoglossum L. Ruta graveolens L. Salix caprea L. Scilla bifolia L. Sedum acre L. Sternbergia colchiciflora Waldst. et Kit. Stipa capillata L. Stipa pulcherrima C. Koch Stipa tirsa Stev. Taxus baccata L. Tilia rubra DC. Valeriana officinalis L. The plants with protection statute, endemites and relicts.../ Ovidius University Annals of Biology-Ecology 14: 25-31 (2010) 80. Vicia pisiformis L. Two species are included in Application II of Directive 92/43/ЕЕС: Cyclamen coum Mill. and Himantoglossum caprinum (Bieb.) C. Koch. Directive 92/43/ЕЕС 60 51 CITES 50 42 IUCN Red List 40 32 30 24 29 Red book BDA 21 20 Herbs prohibited from collecting 12 10 6 Herbs in the restrictive regime 2 Endemites 0 1 Relicts Fig. 1. Proportion of species with conservation status, endemites and relicts In the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) are included 24 species: Anacamptis pyramidalis C. Rich., Cephalanthera damasonium (Mill.) Druce, Cephalanthera longifolia (L.) Fritsch, Cephalanthera rubra (L.) Rich., Cyclamen coum Mill., Dactylorhiza saccifera (Brongn.) Soo, Epipactis helleborine (L.) Crantz, Epipactis microphylla (Ehrh.) Sw., Epipactis purpurata Smith, Galanthus elwesii Hook. fil., Galanthus nivalis L., Himantoglossum caprinum (Bieb.) C. Koch, Limodorum abortivum (L.) Sw., Listera ovata (L.) R. Br., Neottia nidus-avis (L.) Rich., Ophrys apifera Huds., Ophrys cornuta Stev., Ophrys mammosa Desf., Orchis morio L., Orchis purpurea Huds., Orchis simia Lam., Orchis tridentata Scop., Platanthera chlorantha (Cust.) Rchb., Sternbergia colchiciflora Waldst. et Kit. The IUCN Red List for Bulgaria are included 32 species. In category „threatened” are included 13 species: Aesculus hippocastanum L., Anthemis regisborisii Stoj. et Acht., Artemisia pedemontana Balb., Campanula euxina (Vel.) Ancev, Celtis glabrata Steven, Epipactis purpurata Smith, Galanthus elwesii Hook. fil., Galanthus nivalis L., Juniperus sabina L., Jurinea ledebourii Bunge, Ophrys apifera Huds., Ruta graveolens L., Taxus baccata L. In category „vulnerable” are included 9 species: Anacamptis pyramidalis C. Rich., Epipactis microphylla (Ehrh.) Sw., Fibigia clypeata (L.) Medic., Haplophyllum 28 thesioides G. Don., Himantoglossum caprinum (Bieb.) C. Koch, Limodorum abortivum (L.) Sw., Ophrys cornuta Stev., Ophrys mammosa Desf., Pastinaca umbrosa Stev. et DC. In category „nearly threatened” 5 species: Anemone sylvestris L., Cercis siliquastrum L., Erodium hoefftianum C. A. Mey., Galium rubioides L., Vicia pisiformis L. In category „least concern” are included 5 species: Aegilops geniculata Roth, Cyclamen coum Mill., Fritillaria pontica Wahl., Pulmonaria mollis Horn., Tilia rubra DC. In the Red book for PR Bulgaria are included total of 21 species. In the category „endangered” are included 7 species: Aesculus hippocastanum L., Anemone sylvestris L., Artemisia pedemontana Balb., Galanthus nivalis L., Galium rubioides L., Ruta graveolens L., Taxus baccata L. In category „rare” are included 14 species: Anthemis regisborisii Stoj. et Acht., Celtis glabrata Steven, Cercis siliquastrum L., Cyclamen coum Mill., Erodium hoefftianum C. A. Mey., Fibigia clypeata (L.) Medic., Fritillaria pontica Wahl., Haplophyllum thesioides G. Don., Juniperus sabina L., Jurinea ledebourii Bunge, Limodorum abortivum (L.) Sw., Pastinaca umbrosa Stev. et DC., Tilia rubra DC., Vicia pisiformis L. In the Biological Diversity Act are included total of 51 species. In the category „protected” (Application 3) are included 23 species: Aesculus hippocastanum L., Anacamptis pyramidalis C. Rich., Anemone sylvestris L., Anthemis regis-borisii Stoj. et Acht., Artemisia pedemontana Balb., Campanula euxina (Vel.) Ancev, Centaurea marshalliana Spreng., Cyclamen coum Mill., Epipactis purpurata Smith, Fritillaria pontica Wahl., Galanthus elwesii Hook. fil., Galanthus nivalis L., Galium rubioides L., Haplophyllum thesioides G. Don., Himantoglossum caprinum (Bieb.) C. Koch, Juniperus sabina L., Jurinea ledebourii Bunge, Limodorum abortivum (L.) Sw., Ophrys apifera Huds., Ophrys cornuta Stev., Ophrys mammosa Desf., Ruta graveolens L., Taxus baccata L. In the category “under protection and under controlled use” (Application 4) are 28 species: Asparagus tenuifolius Lam., Asparagus verticillatus L., Bupleurum affine Sadl., Bupleurum apiculatum Friv., Bupleurum praealtum L., Bupleurum rotundifolium L., Crocus flavus West., Crocus pallasii Bieb., Dactylorhiza saccifera (Brongn.) Soo, Dimcho Zahariev, Elka Radoslavova / Ovidius University Annals of Biology-Ecology 14: 25-31 (2010) Dryopteris filix-mas (L.) Schott, Echinops sphaerocephalos L., Gypsophila paniculata L., Helichrysum arenarium (L.) Mornh., Lilium martagon L., Orchis morio L., Orchis purpurea Huds., Orchis simia Lam., Orchis tridentata Scop., Polygonatum odoratum (Mill.) Druce, Polystichum aculeatum (L.) Roth, Primula veris L., Ruscus aculeatus L., Ruscus hypoglossum L., Salix caprea L., Scilla bifolia L., Stipa capillata L., Stipa pulcherrima C. Koch, Stipa tirsa Stev. Prohibited is the collecting ofherbs from the natural habitats of 12 species: Althaea officinalis L., Asarum europaeum L., Asplenium trichomanes L., Convallaria majalis L., Helichrysum arenarium (L.) Mornh., Orchis morio L., Orchis purpurea Huds., Orchis simia Lam., Orchis tridentata Scop., Phyllitis scolopendrium (L.) Newm., Ruscus aculeatus L., Valeriana officinalis L. Under a controlled use are 6 species: Berberis vulgaris L., Betonica officinalis L., Carlina acanthifolia All., Galium odoratum (L.) Scop., Primula veris L., Sedum acre L. Endemic species (Figure 1) are relatively well represented – 29 species (3.69% of all species on the plateau). Their number is close to the nation-wide average – 4.86% [24]. This group includes 17 Balkan subendemites: Campanula grossekii Heuff., Campanula lingulata W. et K., Carduus candicans Waldst. et Kit., Chaerophyllum byzantinum Boiss., Doronicum orientale Hoffm., Galium heldreichii Hal., Galium paschale Forsskal, Galium pseudoaristatum Schur., Ophrys cornuta Stev., Pseudolysimachion barrelieri (Schott ex Roem. et Schult.) Holub, Salvia amplexicaulis Lam., Senecio papposus (Reichenb.) Less., Stachys obliqua Waldst. et Kit., Symphytum ottomanum Friv., Syringa vulgaris L., Thesium simplex Vel., Verbascum lychnitis L. The Balkan endemites are 9 species: Achillea clypeolata Sibth. et Sm., Aesculus hippocastanum L., Bupleurum apiculatum Friv., Inula aschersoniana Janka, Knautia macedonica Griseb., Koeleria simonkaii Adam., Onosma thracica Vel., Salvia ringens Sibth. et Sm., Sesleria latifolia (Adam.) Deg. The Bulgarian endemites are 3 species: Anthemis regis-borisii Stoj. et Acht., Campanula euxina (Vel.) Ancev, Myosotis aspera Vel. Data for the relict species on the area of the plateau was first published by Zahariev and Radoslavova [14]. The flora of the plateau included 29 significant number of relict species – 42 (Figure 1). They account for 5.34% of the total species. The majority of them, 39 species, are Tertiary relicts: Abies alba Mil., Acer campestre L., Acer hyrcanum Fisch. et C. A. Meyer, Acer pseudoplatanus L., Acer tataricum L., Aesculus hippocastanum L., Betula pendula Roth, Carpinus betulus L., Carpinus orientalis Mill., Celtis glabrata Steven, Cercis siliquastrum L., Clematis vitalba L., Corylus avellana L., Cotinus coggygria Scop., Cyclamen coum Mill., Fraxinus excelsior L., Fraxinus ornus L., Hedera helix L., Juniperus communis L., Lathyrus aureus (Stev.) Brandza, Pastinaca umbrosa Stev. et DС., Phragmites australis (Cav.) Steud., Picea abies (L.) Karsten, Pinus nigra Arn., Populus alba L., Populus nigra L., Populus tremula L., Pteridium aquilinum (L.) Kuhn., Quercus cerris L., Quercus dalechampii Ten., Ruscus aculeatus L., Ruscus hypoglossum L., Salix alba L., Salix caprea L., Taxus baccata L., Ulmus laevis Pall., Ulmus minor Mill., Viburnum lantana L., Viscum album L. They were widespread during the Tertiary, but their habitats today are much smaller. The second group are quaternary relicts. They have become part of our flora as a result of glaciation during the Quaternary. Therefore, they are considered glacial relicts. On the plateau, there are two such species: Limodorum abortivum (L.) Sw. and Galanthus nivalis L. From the third group, the postglacial steppe relict, only one species is found: Sternbergia colchiciflora Waldst. et Kit. The species with highest conservation value, i.e. those that fall into the categories of being endangered and vulnerable, are 24 in number. With the highest conservation value is Cyclamen coum Mill., which is included in 6 different lists of endangered species: Directive 92/43/ЕЕС, CITES, IUCN Red List, Red book, BDA, Tertiary relicts. Second comes the group of the species Galanthus nivalis L. and Limodorum abortivum (L.) Sw. They appear in 5 different lists: CITES, IUCN Red List, Red book, BDA, quaternary relicts. This also applies to Aesculus hippocastanum L., which is included in the following lists: IUCN Red List, Red book, BDA, Balkan endemites, Tertiary relicts. The third group of species that is listed in 4 lists is Himantoglossum caprinum (Bieb.) C. Koch. (Directive 92/43/ЕЕС, CITES, IUCN Red List, ЗБР), Anthemis regis-borisii Stoj. et Acht. (IUCN Red List, The plants with protection statute, endemites and relicts.../ Ovidius University Annals of Biology-Ecology 14: 25-31 (2010) Red book, BDA, Bulgarian endemites), Ophrys cornuta Stev. (CITES, IUCN Red List, BDA, Balkan subendemites), Taxus baccata L. (IUCN Red List, Red book, BDA, Tertiary relicts). The largest is the group of species that appear in the following lists: • CITES, IUCN Red List, BDA – Anacamptis pyramidalis C. Rich., Epipactis purpurata Smith, Galanthus elwesii Hook. fil., Ophrys apifera Huds., Ophrys mammosa Desf.; • IUCN Red List, Red book, BDA – Anemone sylvestris L., Fritillaria pontica Wahl., Galium rubioides L., Haplophyllum thesioides G. Don., Juniperus sabina L., Jurinea ledebourii Bunge, Ruta graveolens L.; • IUCN Red List, Red book, Tertiary relicts – Celtis glabrata Steven, Cercis siliquastrum L., Pastinaca umbrosa Stev. et DC.; • IUCN Red List, BDA, Bulgarian endemites – Campanula euxina (Vel.) Ancev. 4. Conclusions The total number of species with conservation statute that we found on the Shoumen plateau is 80 (10.18% of all species on the plateau). It is significantly larger than the data published by other authors. In our study, we use more recent documents on nature conservation. They total 6 in comparison to 3 or 4 in previous publications. The species that we described generally appear in 12 lists of endangered species. The endemic species that we found on the plateau and described are 29 species (3.69% of the total number of species). They include 17 Balkan subendemites, 9 Balkan endemites and 3 Bulgarian endemites. The flora of the plateau includes significant number of relict species – 42 (5.34% of the total number of species). The majority of them are Tertiary relicts: 39 species, 2 are quaternary relicts and 1 is postglacial steppe relict. The largest number of species of conservation statute confirms the importance of the Shoumen Plateau as a protected site, preserving the wellbeing of nature in the future. 30 5. References [1] Protected Areas Act, State Gazette number 133 from 11 November 1998, Amended in State Gazette number 98 from 12 November 1999,..., Amended in State Gazette number 19 from 13 March 2009. [2] ANDREEV, N., 1992. Botanical characteristics of National Park Shumensko Plateau, in National Park Shumensko Plateau. Technical Project Green Construction, Agrolesproject, pp. 17–62. [3] Council Directive 92/43/EEC of the European Community to protect natural habitats and of wild fauna and flora. [4] STANEV, S., 2001. Little known names from Bulgarian botany, Pensoft, Sofia – Moscow, 202 pp. [5] VELENOVSKY, J., 1891. Flora Bulgarica, Praga, 676 рp. [6] VELENOVSKY, J., 1898. Flora Bulgarica, Supplementum I, Praga, 420 рp. [7] DAVIDOV, B., 1904. Contribution to study the flora of the district of Shumen, Sbornik ot narodni umotvoreniya, ХХ (II): 1–54. [8] STOIANOV, N., Stefanov B., 1924-1925. Flora of Bulgaria, Vol. I-II, Sofia, pp. 1367. [9] STOIANOV, N., Stefanov B., 1932-1933. Flora of Bulgaria, Vol. I-II, Sofia. [10] STOIANOV, N., Stefanov B., 1947-1948. Flora of Bulgaria, Vol. I-II, Sofia, pp. 1361. [11] STOIANOV, N., Stefanov B., Kitanov, B., 1966-1967. Flora of Bulgaria, Vol. I-II, Nauka i izkustvo, Sofia, pp. 1325. [12] Flora of PR Bulgaria, Vol. І-Х, 1963-1995, Publishing House of BAS, Sofia. [13] RADOSLAVOVA, Е., 2002. The Orchids of the Shumensko Plateau, Snejanka Petkova – AR, Shumen, pp. 48. [14] ZAHARIEV, D., Radoslavova, E., 2010. The Plants of the Shumensko Plateau, Himera, Shumen, pp. 597. [15] Forest development project of the Shumen State Forestry, district Shumen, Vol. I, 2002, Anemone Ltd., Sofia, pp. 148. [16] Red book of PR Bulgaria, Vol. 1, Plants, 1984, Publishing House of BAS, Sofia, 447 pp. [17] Biological Diversity Act, State Gazette number 77 from 9 august 2002, pp. 9–42. Amended in Dimcho Zahariev, Elka Radoslavova / Ovidius University Annals of Biology-Ecology 14: 25-31 (2010) State Gazette number 94 from 16 November 2007. [18] BESHKOV, V. et all.. (eds.), 1994. The Red book of the district Shumen, Slavcho Nikolov & co, Shumen, pp. 199. [19] CHASE, M. (corresponding author), 2003. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II, The Linnean Society of London, Botanical Journal of the Linnean Society, 141: 399–436. [20] ASIOV B., Petrova A., Dimitrov D., Vasilev R., 2006. Conspectus of the Bulgarian vascular flora. Distribution maps and floristic elements, Bulgarian Biodiversity Foundation, Sofia, 452 pp. [21] GRUEV, B., Kuzmanov B., 1994. General biogeography, University Press St. Kliment Ohridski, Sofia, 498 pp. [22] PEEV, D., 2001. National park Rila. Management plan 2001, 2010. Adopted by Resolution №522 of Council of Ministers on 04.07.2001, Sofia, 338 pp. [23] BOŽA, P., Anačkov G., Igić R., Vukov D., Polić D., 2005. Flora “Rimskog šanca” (Vojvodina, Srbija), 8th Symposium on the flora of Southeastern Serbia and Neighbouring Regions, Niš, 20-24.06.2005, Abstracts, рр. 55. [24] PEEV, D., Kozuharov S., Anchev M., Petrova A., Ivanova D., Tzoneva S., 1998. Biodiversity of Vascular Plants in Bulgaria, In: Curt Meine (ed.), Bulgaria's Biological Diversity: Conservation Status and Needs Assessment, Volumes I and II, Washington, D.C., Biodiversity Support Program, pp. 55–88. [25] Convention on International Trade in Endangered Species of Wild Fauna and Flora, State Gazette number 6 from 21 Januari 1992. [26] PETROVA А., Vladimirov V. (eds.), 2009. Red List of Bulgarian vascular plants, Phytologia Balcanica 15 (1): 63–94. [27] Order number RD-72 from 3 februari 2006 for special arrangements for the conservation and use of medicinal plants, State Gazette number 16 from 21 Februari 2006. 31 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 A CHARACTERISTIC OF MODEL HABITATS IN SOUTH DOBRUDJA Dimcho ZAHARIEV University of Shumen Bishop Konstantin Preslavski, Faculty of Nature Sciences, 115 Universitetska Str., 9712, Shumen, Bulgaria, dimtchoz@yahoo.com __________________________________________________________________________________________ Abstract: Five natural habitats and five artificial habitats (forest shelter belts) are investigated in South Dobrudja. Most taxonomical diversity and most protected species from natural habitats are established in Western Pontic Paeonian steppes near to Bejanovo village. In the forest shelter belts is typical less taxonomical diversity, less protected species and more anthropophytes, which due to strong anthropogenically influence. The families with most of genera and species are: Asteraceae, Рoaceae, Rosaceae and Lamiaceae. The biggest groups from biological types are perennial herbaceous plants and annual herbaceous plants. The floristic elements are presented mainly from circumboreal, European and Mediterranean elements. The mainly reasons about high number of anthropophytes are intensive fragmentation of the natural habitats, all round from agricultural areas – a source of anthropophytes, and their accessibility for peoples and domestic animals. Keywords: Dobrudja, habitats, taxonomical diversity, biological types, floristic elements, endemites, relict species, protected species, anthropophytes. __________________________________________________________________________________________ 1. Introduction Dobrudja is historical and geographical area between the lower reaches of the Danube and Black Sea. The area is 23 000 km2. It is divided into two parts – North and South Dobrudja. North Dobrudja is located in Southeastern Romania. Its area covers about 2/3 of the territory, amounting to 15 435 km². South Dobrudja is located in Northeastern Bulgaria. The area is 7 565 km². The Bulgarian part of Dobrudja is divided by the virtual line between Stojer village and Rosica village into two parts – eastern and western. South Dobrudja is located in 3 administrative areas – Varnenska (municipality Aksakovo), Dobrichka (all municipalities) and Silistrenska (municipality Kainardja). The climate is temperate. It is characterized by warm summers and cold winters, high annual amplitude of air temperature, spring–summer minimum and winter maximum of rainfall, the snow cover is relatively stable. The average temperatures in January are between 0°С and –1.5°С. In the summer dominated tropical and subtropical air masses and the average temperature in July is 22-24°С. The spring ISSN-1453-1267 and the autumn are approximately the same temperatures. April was warmer in October. The rainfalls are with maximum in May–June and with minimum in February–March. The annual amount of precipitation is 520 to 650 mm. About 10% of the total amount of precipitation is snow [1]. In South Dobrudja dominated haplic Chernozems. Small areas are covered with kastanic, calcaric or gleyc Chernozems. On the coast of the Black Sea and the rivers are distributed rendzic Leptosols and Nitisols. Along the Danube are distributed calcaric Fluvisols, Histosols and Gleysols. Unique to the region are the small in area Vertisols [2]. In terms of its flora, South Dobrudja belongs to the region of Northeastern Bulgaria. On its territory are described 1 508 species, which are referred to 496 genera and 144 families. Natural vegetation was composed of forest steppes, which include large forest complexes and grasslands. Original natural vegetation of the Dobrudja was destroyed in a large part, due to intensive human activities [3]. Today on the territory of Southern Dobrudja occur 41 different plant communities – primary and secondary [4]. 33 habitats are described according to Council Directive 92/43/EEC of the European Community to protect © 2010 Ovidius University Press A characteristic of model habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) natural habitats and of wild fauna and flora [5, 6, 7, 8, 9, 10, 11, 12, 13, 14]. anthropophytes are presented by Stefanov and Kitanov [32]. is recorded by the system of effects 2. Material and Methods used in the assessment of an object from the network of protected areas Nature 2000. The field studies were conducted on the route method in 2008 – 2009. Subject of research are a total of 10 different habitats – 5 natural and 5 artificial. The natural habitats are defined by Kavrakova et all. [15]. Each habitat is characterized as follows: average altitude, exposure, slope, area, soil type and subtype, base rock, cover of tree, shrub and herbaceous vegetation, number of established species, genera and families, cover of each species, distribution in biological type, floristic elements, endemites, subendemites and relict species, species with conservation status, anthropophytes, anthropogenic influence. The average altitude, exposure, slope and area are defined with map at a scale 1:50 000. The soil types and subtypes are presented by Ninov [2]. The taxons and the biological type are defined by Identifier of the vascular plants in Bulgatia [16], Flora of PR Bulgaria, Vol. І – Х [17]. The update of the taxons is consistent with APG II [18] and Petrova et all. [19]. The cover of each species is presented by Braun-Blanquet [20]. The following symbols are used: r – cover less than 5%, one individual; + – cover less than 5%, 2-5 individuals; 1 – cover less than 5%, 6-50 individuals; 2m – cover less than 5%, more than 50 individuals; 2a – cover 5-12.5%; 2b – cover 12.5-25%; 3 – cover 25-50%; 4 – cover 50-75%; 5 – cover 75-100%. The following symbols are used [16] for biological types: t (from English tree), sh (from English shrub), p (from English perennial), а (from English annual). The floristic elements, endemites and subendemites are presented by Asiov et all. [21]. The relicts are presented by Gruev and Kuzmanov [22], Peev [23], Boža et all. [24], Peev et all. [25]. The conservation status is presented using the following documents: Council Directive 92/43/EEC of the European Community to protect natural habitats and of wild fauna and flora [26], Berne Convention [27], Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) [28], Red book of PR Bulgaria [29], IUCN Red List for Bulgaria [30], Biological Diversity Act [27], Order for special arrangements for the conservation and use of medicinal plants [31]. The 3. Results and Discussion HABITAT 1 A habitat by Nature 2000: Euro-Siberian steppic woods with Quercus spp. A habitat by Bondev [4]: Cerris oak (Querceta cerris) forests. It is located southwest of Efreitor Bakalovo village, municipality Krushari. The territory is a part of Nature 2000 (Protected area “Suha reka”). The average altitude is 150 m. The exposure is south. The slope varies in different parts. It is smaller in the north and higher in southern parts. The area of the habitat is 6 300 dka. The soil type is Chernozems, and the soil subtype is haplic Chernozems. The bedrock is limestone. The cover of the tree vegetation is 80%, the cover of the shrub vegetation is 10% and the cover of the herbaceous vegetation is 10%. In the habitat have been indetified 78 species of vascular plants from 67 genera and 27 families. The families with greatest number of genera are as follows: Asteraceae – 8 (11.94%), Poaceae – 8 (11.94%), Brassicaceae – 5 (7.46%) and Fabaceae – 5 (7.46%). The families with greatest number of species are as follows: Poaceae – 10 (12.82%), Rosaceae – 9 (11.54%), Asteraceae – 8 (10.26%), Brassicaceae – 6 (7.69%), Fabaceae – 5 (6.41%), Lamiaceae – 5 (6.41%) and Scrophulariaceae – 5 (6.41%). The genera with greatest number of species are as follows: Veronica – with 4 species (5.13%), Poa – with 3 species (3.85%) and Potentilla – with 3 species (3.85%). With the highest percentage of coverage are Quercus cerris L. (5) and Poa nemoralis L. (2a). With the lowest percentage of coverage (1) are Cornus mas L., Carduus nutans L. and Vicia sativa L. Each of the remaining 73 species has coverage 2m. The distribution of species in biological type is as follows: The perennial herbaceous plants (p) are most – they are 38 species (48.72%). Secondly, are 34 Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) annual herbaceous plants (a) with 21 species (26.92%). The next is the transition group of annual and reaches 30°. The area of the habitat is 400 dka. The soil type is Chernozems, and the soil subtype is haplic Chernozems. The bedrock is limestone. The cover of the shrub vegetation is less to biennial herbaceous plants (a-b) with 6 species (7.69%). The trees (t) and the transition group of shrubs to trees (sh-t) have equal number of species – 4 (5.13%). The biennial herbaceous plants (b) are 3 species (3.85%), and shrubs (sh) are 2 species (2.56%) only. The diversity of floristic elements is as follows: The largest number of species (28) has circumboreal origin. The next are species with European origin – they are 25 species. 13 species have Mediterranean origin. The Pontic type of elements and cosmopolitans are 5 species each of them. One of the species is adventive element. One of the species is Balkan subendemite – Ornithogalum sibthorpii Greut. Three species are Tertiary relicts: Carpinus orientalis Mill., Quercus cerris L. and Ulmus minor Mill. Two species with protection statute are established – Crocus flavus West. and Scilla bifolia L. They are included in the Biological Diversity Act in the category „Under the protection and regulated use of nature”. The anthropophytes are 55 species (70.51%). The large number indicates for increased anthropogenic impact on the habitat. The anthropogenic influence consists in the following: 1. Forestry felling. 2. Artificial afforestation. 3. Grazing sheep, goats and cows. 4. Pollution by garbage from the shepherds and farm workers. 5. Arable land in the vicinity. 6. Improved access to the habitat by a system of paths and roads. 7. Тourist pavilion with a fireplace. 8. Fountain with several troughs. than 5% and the cover of the herbaceous vegetation is 90%. More than 5% of the ground is devoid of vegetation cover. In the habitat have been indetified 83 species of vascular plants from 70 genera and 24 families. The families with greatest number of genera are as follows: Rosaceae – 10 (14.29%), Asteraceae – 7 (10.00%), Lamiaceae – 7 (10.00%), Poaceae – 7 (10.00%) and Apiaceae – 5 (7.14%). The families with greatest number of species are as follows: Rosaceae – 10 (12.05%), Lamiaceae – 10 (12.05%), Asteraceae – 9 (10.84%), Poaceae – 7 (8.43%), Apiaceae – 5 (6.02%), Caryophillaceae – 5 (6.02%) and Ranunculaceae – 5 (6.02%). The genera with greatest number of species are as follows: Euphorbia and Salvia – with 3 species each of them (3.61%). Stipa capillata L. (2а) is with the highest percentage of coverage. With the lowest percentage of coverage are Robinia pseudoacacia L. (1), Althaea cannabina L. (1), Prunus mahaleb L. (+), Carduus nutans L. (+), Pyrus pyraster Burgsd. (+), Ligustrum vulgare L. (r) and Malus sylvestris Mill. (r). From the neighboring shelter belt immigrated some tree species. The reason for this is the transference of fruits and seeds by birds and wind. Each of the remaining 75 species has coverage 2m. The distribution of species in biological type is as follows: The perennial herbaceous plants (p) are most – they are 48 species (57.83%). Secondly, are annual herbaceous plants (a) with 18 species (21.69%). The biennial herbaceous plants (b) and the trees have 4 species each of them (4.82%). The shrubs (sh) are 3 species (3.61%). The transition group of shrubs to trees (sh-t) and the transition group of annual to perennial herbaceous plants (a-р) have 2 species each of them (2.41%). The transition groups of annual to biennial herbaceous plants (a-b) and of biennial to perennial herbaceous plants (b-р) have one species (1.20%) each of them. The most species are species with Mediterranean (23 species), European (22 species) and circumboreal origin (20 species). The Pontic type of elements are 10 species. The cosmopolitans are 3 species. The Balkan endemites are 2 species – Achillea clypeolata HABITAT 2 A habitat by Bondev [4]: Shrub (Amygdaleta nanae) and grass (Artemisieta albae, Agropyreta pectiniformae, Agropyreta brandzae, Brometa riparii etc.) steppe and xerothermal communities. It is located south of Karapelit village, municipality Dobrich. The territory is a part of Nature 2000 (Protected area “Suha reka”). The average altitude is 160 m. The exposure is in some parts south, while in others – west. The slope is variable 35 A characteristic of model habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) Sibth. et Sm. and Salvia ringens Sibth. et Sm. One species is Balkan subendemite – Dianthus pallens Sm. One species has adventive origin and one species has Alpine-Mediterranean. Brassicaceae – 5 (5.95%). The families with greatest number of species are as follows: Poaceae – 14 (13.59%), Lamiaceae – 13 (12.62%), Asteraceae – 12 (11.65%), Brassicaceae – 5 (4.85%), Euphorbiaceae – 5 (4.85%) and Rubiaceae – 5 (4.85%). The genus Four species with protection statute are established: Adonis vernalis L. is included in CITES and in the Order for special arrangements for the conservation and use of medicinal plants in the category “Collecting herbs is prohibited from the natural habitats”. Jurinea ledebourii Bunge is included in the IUCN Red List for Bulgaria in the category “Endangered”, in the Red book for Bulgaria in the category „Rare” and in the Biological Diversity Act in the category „Protected”. Two species are included in the Biological Diversity Act in the category „Under the protection and regulated use of nature”: Bupleurum affine Sadl. and Stipa capillata L. The anthropophytes are 53 species (63.86%). They are an indicator of the extent of human impact on habitat. The anthropogenic influence on the habitat due to the presence of: 1. Improved access to the habitat by a system roads. 2. Arable land in the vicinity. 3. The forest shelter belts in the vicinity. Euphorbia is with greatest number of species – with 5 species (4.85%). With the highest percentage of coverage are Poa pratensis L. (2b) and Elymus repens (L.) Gould. (2а). With the lowest percentage of coverage (1) is Carduus thoermeri Weinm. Each of the remaining 100 species has coverage 2m. The distribution of species in biological type is as follows: The perennial herbaceous plants (p) are most – they are 53 species (51.46%). Secondly, are annual herbaceous plants (a) with 32 species (31.07%). The biennial herbaceous plants (b) are 10 species (9.71%). The transition group of annual to biennial herbaceous plants (a-b) has 3 species (2.91%). The transition group of annual to perennial herbaceous plants (a-р) has 2 species (1.94%). The trees (t) are 2 species (1.94%) and the shrubs (sh) – one species (3.61%) only. The largest number of species (31) has circumboreal origin. Secondly, are European (27 species) and Mediterranean type of elements (20 species). The species with Pontic origin are 13. The cosmopolitans are 6 species. Three species are Balkan subendemites – Ornithogalum sibthorpii Greut., Verbascum banaticum Schrad. and Carduus thoermeri Weinm. The adventive species are 2. One species has Alpine-Carpathian origin. Three species with protection statute are established: Artemisia pedemontana Balb. is included in IUCN Red List for Bulgaria in the category „Endangered”, in the Red book for Bulgaria in the category „Threatened with extinction” and in the Biological Diversity Act in the category „Protected”. Helichrysum arenarium (L.) Mornh. and Stipa capillata L. are included in the Biological Diversity Act in the category “Under the protection and regulated use of nature”. Helichrysum arenarium (L.) Mornh. is included in the Order for special arrangements for the conservation and use of medicinal plants in the category “Collecting herbs is prohibited from the natural habitats”. HABITAT 3 A habitat by Bondev [4]: Mesoxerothermal grass vegetation with a prevalence of Poa bulbosa L., Lolium perenne L., Cynodon dactylon L., partly also Dichantium ischaemum (L.) Roberty and rarely Chrysopogon gryllus (L.) Tryn. It is located between Izvorovo and Krasen villages, municipality General Toshevo. The average altitude is 180 m. The exposure is southwest. The slope is variable and reaches 30°. The area of the habitat is 2 000 dka. The soil type is Leptosols, and the soil subtype is rendzic Leptosols. The bedrock is limestone. The cover of the shrub vegetation is less than 5% and the cover of the herbaceous vegetation is 80%. More than 15% of the ground is devoid of vegetation cover. In the habitat have been indetified 103 species of vascular plants from 84 genera and 32 families. The families with greatest number of genera are as follows: Asteraceae – 15 (14.56%), Poaceae – 12 (14.29%), Lamiaceae – 10 (11.90%) and 36 Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) The anthropophytes are 80 species (77.67%). They show a significant anthropogenic impact on the habitat. The anthropogenic influence on the habitat due to the presence of: 1. Improved access to the habitat by a system roads. 2. Arable land in the vicinity. 3. Transmission line, passing through the territory. 4. Grazing sheep and goats. 5. Pollution by garbage L. and Stipa capillata L. With the cover 1 are 14 species. With the lowest percentage of coverage are Malus dasyphylla Borkh. (+) and Cydonia oblonga Mill. (r). They are most likely carried by birds. Each of the remaining 134 species has coverage 2m. The distribution of species in biological type is as follows: The perennial herbaceous plants (p) are most – they are 88 species (57.52%). Secondly, are from the two villages. 6. Artificial terracing of slopes. annual herbaceous plants (a) with 38 species (24.84%). The next is the transition group of annual to biennial herbaceous plants (a-b) with 7 species (4.58%). The trees (t) and the shrubs (sh) are 5 species each of them (3.27%). The biennial herbaceous plants (b) are 4 species (2.61%). The transition groups of annual to perennial herbaceous plants (a-р), of biennial to perennial herbaceous plants (b-p) and of shrubs to trees (sh-t) have 2 species each of them (1.31%). The largest number of species (39) has circumboreal origin. The next are species with Mediterranean and European type of elements – with 37 species each of them. Thirdly, are the Pontic type of elements with 21 species. The cosmopolitan are 6 species. Four of the species are Balkan endemites – Achillea clypeolata Sibth. et Sm., Astragalus spruneri Boiss., Chamaecytisus jankae (Vel.) Rothm. and Potentilla emili-popii Nyar. Five of the species are Balkan subendemites – Carduus thoermeri Weinm., Centaurea napulifera Roch., Dianthus pallens Sm., Ornithogalum sibthorpii Greut. and Thesium simplex Vel. The remaining 3 species have Alpine-Mediterranean, Oriental-Turanian and Pannonian-Pontic origin. Eight species with protection statute are established: Paeonia tenuifolia L. is included in Berne Convention, in Directive 92/43/ЕЕС and in the Biological Diversity Act in the category “Protected”. Potentilla emili-popii Nyar. is included in Berne Convention, in Directive 92/43/ЕЕС, in the Biological Diversity Act in the category „Declaration of protected areas to protect habitat for species by Directive 92/43/ЕEC” and in the category „Protected”. Adonis vernalis L. is included in CITES and in the Order for special arrangements for the conservation and use of medicinal plants in the category “Collecting herbs is prohibited from the natural habitats”. Artemisia pedemontana Balb. is included in IUCN Red List for Bulgaria in the HABITAT 4 A habitat by Nature 2000: Western Pontic Paeonian steppes It is located near Bejanovo village, municipality General Toshevo. The territory is a part of Nature 2000 (Protected area “Kraimorska Dobrudja”). The average altitude is 80 m. The exposure is northeast. The slope is low and reaches 5°. The area of the habitat is 650 dka. The soil type is Chernozems, and the soil subtype is calcaric Chernozems. The bedrock is limestone. The cover of the shrub vegetation is less than 5% and the cover of the herbaceous vegetation is 70%. More than 25% of the ground is devoid of vegetation cover. In the habitat have been indetified 153 species of vascular plants from 116 genera and 36 families. It is the richest of plant species from the natural habitats. The families with greatest number of genera are as follows: Asteraceae – 13 (11.21%), Rosaceae – 12 (10.34%), Lamiaceae – 11 (9.48%), Poaceae – 11 (9.48%), Boraginaceae – 6 (5.17%), Brassicaceae – 6 (5.17%), Fabaceae – 6 (5.17%), Apiaceae – 5 (4.31%), Ranunculaceae – 5 (4.31%) and Scrophulariaceae – 5 (4.31%). The families with greatest number of species are as follows: Asteraceae – 18 (11.76%), Rosaceae – 17 (11.11%), Lamiaceae – 15 (9.80%), Poaceae – 14 (9.15%), Boraginaceae – 8 (5.23%), Caryophyllaceae – 8 (5.23%), Fabaceae – 7 (4.58%), Apiaceae – 6 (3.92%), Brassicaceae – 6 (3.92%), Ranunculaceae – 6 (3.92%), Euphorbiaceae – 5 (3.27%), Rubiaceae – 5 (3.27%) and Scrophulariaceae – 5 (3.27%). The genera with greatest number of species are as follows: Euphorbia – with 5 species (3.27%), Cerastium, Potentilla, Prunus, Salvia and Silene – with 3 species each of them (1.96%). With the highest percentage of coverage (2b) are Festuca pseudovina Hack. ex Wiesd., Poa pratensis 37 A characteristic of model habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) With the highest percentage of coverage (2а) is Dichantium ischaemum (L.) Roberty. With the lowest percentage of coverage (+) are Gleditsia triacanthos L., Cornus sanguinea L. and Prunus spinosa L. Each of the remaining 42 species has coverage 2m. The perennial herbaceous plants (p) are most – they are 24 species (52.17%). Secondly, are annual herbaceous plants (a) with 8 species (17.39%). The shrubs are 6 species (13.04%). The biennial herbaceous plants (b) are 3 species (6.52%). The category „Endangered”, in the Red book for Bulgaria in the category „Threatened with extinction” and in the Biological Diversity Act in the category „Protected”. Erodium hoefftianum C. A. Mey. is included in the Red book for Bulgaria in the category „Rare” and in IUCN Red List for Bulgaria in the category „Near Threatened”. Pulsatilla montana (Hoppe) Reichenb., Stipa capillata L. and Stipa lessingiana Trin. et Rupr. are included in the Biological Diversity Act in the category “Under the protection and regulated use of nature”. The anthropophytes are 92 species (60.13%), which indicates a high anthropogenic impact on the habitat. The anthropogenic influence on the habitat due to the presence of: 1. Improved access to the habitat by a system roads. 2. Arable land in the vicinity. 3. The forest shelter belts and artificial forest from Robinia pseudoacacia L. in the vicinity. 4. Grazing cows. 5. Disposal of soil in the vicinity. transition group of shrubs to trees (sh-t) has 2 species (4.35%). The trees (t), the transition groups of annual to biennial herbaceous plants (а-b) and of annual to perennial herbaceous plants (a-р) have one species (2.17%) each of them. The largest number of species (14) has Mediterranean origin. Secondly, are circumboreal type of elements with 11 species. The next are species with Pontic (9 species) and European origin (8 species). The cosmopolitan are 3 species. One of the species is adventive element. Two species with protection statute are established: Stipa capillata L. is included in the Biological Diversity Act in the category „Under the protection and regulated use of nature”. Sedum acre L. is included in the Order for special arrangements for the conservation and use of medicinal plants in the category “Under a restrictive regime”. The anthropophytes are 32 species (69.57%). The high rate is due to human activities in adjacent areas of the habitat. The anthropogenic influence on the habitat due to the presence of: 1. Improved access to the habitat by a system roads. 2. Grazing cows in the bottom of the slope. 3. Arable land in the vicinity. HABITAT 5 A habitat by Nature 2000: Rupicolous calcareous or basophilic grasslands of the AlyssoSedion albi. It is located between Onogur and Efreitor Bakalovo villages, municipality Krushari. The territory is a part of Nature 2000 (Protected area “Suha reka”). The average altitude is 70 m. The exposure is south. The slope is variable and reaches 40°. The area of the habitat is 30 dka. The soil type is Leptosols, and the soil subtype is rendzic Leptosols. The bedrock is limestone. The cover of the shrub vegetation is less than 5% and the cover of the herbaceous vegetation is 30%. More than 65% of the ground is devoid of vegetation cover. In the habitat have been indetified 46 species of vascular plants from 43 genera and 22 families. It is the most poor of plant species from the natural habitats. The families with greatest number of genera are as follows: Asteraceae – 7 (16.28%), Рoaceae – 5 (11.63%), Lamiaceae and Apiaceae – with 4 species each of them (9.30%). The families with greatest number of species are as follows: Asteraceae – 8 (17.39%), Lamiaceae and Poaceae – with 5 species each of them (10.87%). The genera with greatest number of species are as follows: Centaurea, Sedum and Teucrium – with 2 species each of them (4.35%). HABITAT 6 Forest shelter belt formed by Quercus cerris L. It is located between General Toshevo and Liuliakovo village, municipality General Toshevo. The average altitude is 210 m. The exposure is west. The shelter belt is oriented in a southwest – northeast direction. The slope is low and reaches 5°. The area is 75 dka. The length of the shelter belt is 5 000 m, and the width – 15 m. The soil type is Chernozems, and the soil subtype is haplic Chernozems. The bedrock is limestone. The cover of the tree vegetation 38 Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) is 80%, the cover of the shrub vegetation is 10% and the cover of the herbaceous vegetation is 60%. In the shelter belt have been indetified 49 species of vascular plants from 44 genera and 21 families. The families with greatest number of genera are as follows: Asteraceae and Рoaceae – with 8 species each of them (18.18%), Rosaceae – 6 (13.64%) and Lamiaceae – 4 (9.09%). The families with greatest number of species are as follows: Asteraceae and Рoaceae – with 8 species each of them (16.33%), Rosaceae – 6 (12.24%) and Lamiaceae – 4 (8.16%). The anthropophytes are 38 species (77.55%). The high number is due to the artificial origin of the habitat and adjacent to farmland. The anthropogenic influence due to the presence of: 1. Improved access to the habitat by a system roads. 2. Grazing goats and cows. 3. Pollution by garbage from the shepherds and farm workers. 4. Arable land in the vicinity. The genera with greatest number of species are as follows: Avenula, Cirsium, Galium, Prunus and Sambucus – with 2 species each of them (4.08%). With the highest percentage of coverage (5) are Quercus cerris L. and Poa pratensis L. (3). With coverage 2b are Avenula compressa (Heuff.) Sauer et Chmelit., Avenula pubescens (Huds.) Dumort. and Lolium perenne L. With coverage 2a are Robinia pseudoacacia L. and Hordeum hystrix Roth. With the lowest percentage of coverage (+) are Crataegus monogyna Jacq., Cirsium arvense (L.) Scop. and Euphorbia agraria Bieb. Only with one individual (r) is Celtis australis L. Each of the remaining 38 species has coverage 2m. The perennial herbaceous plants (p) are most – they are 34 species (69.39%). Secondly, are annual herbaceous plants (a) with 10 species (20.41%). Thirdly, are the shrubs (sh) with 5 species (10.20%). The trees (t) are 4 species (8.16%). The transition group of shrubs to trees (sh-t) has 3 species (6.12%). The biennial herbaceous plants (b) are 2 species (4.08%). The transition group of annual to biennial herbaceous plants (а-b) has 1 species (2.04%) only. The largest number of species (20) has circumboreal origin. Secondly, are species with European (11) and Mediterranean origin (10). The cosmopolitan are 3 species. Two of the species are adventive elements. One of the species has Pontic origin. One of the species is Balkan subendemite – Galium pseudoaristatum Schur. One species is Tertiary relict in all of the habitat – Quercus cerris L. It is wooded artificial for the creation of the shelter belt. There are no species of protection status. This can be explained easily by the artificial origin of the habitat. L. HABITAT 7 Forest shelter belt formed by Fraxinus excelsior It is located near Chernookovo village, municipality General Toshevo. The average altitude is 160 m. The exposure is east. The shelter belt is oriented in a southwest – northeast direction. The slope is low and reaches 5°. The area is 31.5 dka. The length of the shelter belt is 2 100 m, the width – 15 m, and the height – 15 m. The soil type is Chernozems, and the soil subtype is haplic Chernozems. The bedrock is limestone. The cover of the tree vegetation is 80%, the cover of the shrub vegetation is 10% and the cover of the herbaceous vegetation is 60%. In the shelter belt have been indetified 59 species of vascular plants from 50 genera and 19 families. The families with greatest number of genera are as follows: Asteraceae – 11 (22.00%), Рosaceae – 6 (12.00%) and Rosaceae – 6 (12.00%). The families with greatest number of species are as follows: Asteraceae – 14 (23.73%), Рosaceae – 8 (13.56%) and Rosaceae – 6 (10.17%). The genera with greatest number of species are as follows: Chenopodium and Fraxinus – with 3 species each of them (5.08%); Artemisia, Bromus, Carduus, Centaurea, Consolida and Hordeum – with 2 species each of them (3.39%). With the highest percentage of coverage (5) is Fraxinus excelsior L., followed by Poa pratensis L. (2а). With the lowest percentage of coverage (+) are Amorpha fruticosa L. and Elaeagnus angustifolia L. Only with one individual (r) is Salvia argentea L. With the cover 1 are 4 species. Each of the remaining 50 species has coverage 2m. The perennial herbaceous plants (p) are most – they are 22 species (37.29%). Secondly, are annual herbaceous plants (a) with 16 species (27.12%). Thirdly, are the trees (t) with 7 species (11.86%). The 39 A characteristic of model habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) shrubs (sh), the biennial herbaceous plants (b) and the transition group of annual to biennial herbaceous plants (а-b) have 4 species each of them (6.78%). The transition group of shrubs to trees (sh-t) has 2 species (3.39%). The largest number of species (23) has circumboreal origin. Secondly, are species with European (13) and Mediterranean origin (8). The cosmopolitan are 6 species. Five of the species have Pontic origin. Four of the species are adventive elements. Four species in the habitat are Tertiary relicts: Acer tataricum L., Cotinus coggygria Scop., Fraxinus excelsior L., Quercus cerris L. The main subtype is eutric Vertisols. The bedrock is limestone. The cover of the tree vegetation is 80%, the cover of the shrub vegetation is 10% and the cover of the herbaceous vegetation is 60%. In the shelter belt have been indetified 55 species of vascular plants from 47 genera and 21 families. The families with greatest number of genera are as follows: Asteraceae – 9 (19.15%), Rosaceae – 8 (17.02%) and Рosaceae – 6 (12.77%). The families with greatest number of species are as follows: Asteraceae – 10 (18.18%), Rosaceae – 10 (18.18%) and Рosaceae – 7 (12.73%). The genera with greatest number of species are as follows: Acer and Prunus – with 3 species each of them (5.45%). species is Fraxinus excelsior L. It is wooded artificial for the creation of the shelter belt. Acer tataricum L. and Cotinus coggygria Scop. have less than 5% coverage and their number is more than 50 individuals. The number of the individuals from Quercus cerris L. is less than 50. Perhaps individuals of these three species have evolved from fruit, carried over from adjacent areas. One species with protection statute is established – Fraxinus pallisiae Wilmott. It is included in IUCN Red List for Bulgaria in the category „Vulnerable”. It has less than 5% coverage, and its number is more than 50 individuals. The most likely reason for its presence in the shelter belt is its planting together with basic species Fraxinus excelsior L. The anthropophytes are 51 species (86.44%). Extremely high number of them due to the artificial origin of the habitat and adjacent to farmland. The anthropogenic influence due to the presence of: 1. Improved access to the habitat by a system roads. 2. Pollution by garbage from the shepherds and farm workers. 3. Arable land in the vicinity. With the highest percentage of coverage (4) is Fraxinus oxycarpa Willd. With the lowest percentage of coverage (1) are Amorpha fruticosa L., Tilia cordata Mill. and Tilia tomentosa Moench. Each of the remaining 51 species has coverage 2m. The perennial herbaceous plants (p) are most – they are 22 species (40.00%). Secondly, are annual herbaceous plants (a) with 11 species (20.00%). Thirdly, are the trees (t) with 9 species (16.36%). The shrubs (sh) and the transition group of shrubs to trees (sh-t) have 4 species each of them (7.27%). The next is the transition group of annual to biennial herbaceous plants (a-b) with 2 species (3.64%). The biennial herbaceous plants (b), the transition groups of annual to perennial herbaceous plants (а-р) and of biennual to perennial herbaceous plants (b-р) are presented with one species (1.82%) only. The largest number of species (25) has circumboreal origin. Secondly, are species with European (13) and Mediterranean origin (7). The cosmopolitan are 4 species. Three of the species have Pontic origin. Three of the species are adventive elements. Three species in the habitat are Tertiary relicts: Acer campestre L., Acer tataricum L., Quercus cerris L. Each of them has less than 5% coverage and their number is more than 50 individuals. The reason for their presence can be traced in the transference of fruit from neighboring areas. There are no species of protection status. This can be explained easily by the artificial origin of the habitat. HABITAT 8 Forest shelter belt formed by Fraxinus oxycarpa Willd. It is located as a third shelter belt between Vladimirovo and Benkovski villages, municipality Dobrich. The average altitude is 230 m. The exposure is northeast. The shelter belt is oriented in a southwest – northeast direction. The slope is low and reaches 5°. The area is 17 dka. The length of the shelter belt is 1 150 m, the width – 15 m, and the height – 15 m. The soil type is Vertisols, and the soil 40 Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) The anthropophytes are 45 species (81.82%). The high number is due to the artificial origin of the habitat and adjacent to farmland. The anthropogenic influence due to the presence of: 1. Improved access to the habitat by a system roads. 2. Pollution by garbage. 3. Arable land in the vicinity. Thirdly, are the trees (t) and the shrubs (sh) with 9 species (10.23%) each of them. The next are the transition group of shrubs to trees (sh-t) and the biennial herbaceous plants (b) with 4 species each of them (4.55%). The transition group of annual to perennial herbaceous plants (а-р) has 2 species (2.27%). The transition groups of annual to biennial herbaceous plants (а-b) and of biennual to perennial herbaceous plants (b-р) are presented with one species (1.14%). The largest number of species (28) has circumboreal origin. Secondly, are species with Mediterranean origin (21). The next are species with European (14) and Pontic origin (12). Six of the species is adventive element. The cosmopolitan are 5 species. One of the species has Oriental-Turanian origin. One of the species is Balkan endemite – Achillea clypeolata Sibth. et Sm. HABITAT 9 Forest shelter belt formed by Gleditsia triacanthos L. It is located south of Karapelit village, municipality Dobrich. The average altitude is 175 m. The exposure is northeast. The shelter belt is oriented in a north – south direction. The slope is low and reaches 10°. The area is 15 dka. The length of the shelter belt is 1 000 m, the width – 15 m, and the height – 15 m. The soil type is Chernozems, and the soil subtype is haplic Chernozems. The bedrock is limestone. The cover of the tree vegetation is 60%, the cover of the shrub vegetation is 10% and the cover of the herbaceous vegetation is 70%. In the shelter belt have been indetified 88 species of vascular plants from 78 genera and 30 families. It is the richest of plant species from the shelter belts. The reason for this is that is located immediately adjacent steppe. From the steppe to the shelter belt migrated a large number of species. The families with greatest number of genera are as follows: Lamiaceae – 11 (14.10%), Fabaceae – 9 (11.54%), Rosaceae – 8 (10.26%), Asteraceae – 7 (8.97%), Poaceae – 6 (7.69%) and Apiaceae – 5 (6.41%). The families with greatest number of species are as follows: Lamiaceae – 13 (14.77%), Rosaceae – 11 (12.50%), Asteraceae – 9 (10.23%), Fabaceae – 9 (10.23%), Poaceae – 6 (6.82%) and Apiaceae – 5 (5.68%). The genus with greatest number of species is Prunus – with 4 species (4.55%). With the highest percentage of coverage (4) is Gleditsia triacanthos L. Secondly, it is Poa pratensis L. (2а). With the cover 1 are 23 species. With the lowest percentage of coverage are Carduus acanthoides L., Sanguisorba minor Scop. (+) and Verbascum ovalifolium Sms. (r). Each of the remaining 60 species has coverage 2m. The perennial herbaceous plants (p) are most – they are 42 species (47.73%). Secondly, are annual herbaceous plants (a) with 16 species (18.18%). Three species are Tertiary relicts: Celtis australis L., Cotinus coggygria Scop., Ulmus minor Mill. Each of them has less than 5% coverage. The number of Celtis australis L. is less than 50 individuals. The number of another two species is more than 50 individuals. The reason for their presence can be traced in the transference of fruit from neighboring areas. Four species with protection statute are established: Adonis vernalis L. is included in CITES and in the Order for special arrangements for the conservation and use of medicinal plants in the category “Collecting herbs is prohibited from the natural habitats”. Jurinea ledebourii Bunge is included in the IUCN Red List for Bulgaria in the category “Endangered”, in the Red book for Bulgaria in the category „rare” and in the Biological Diversity Act in the category „protected”. Tilia rubra DC. is included in IUCN Red List for Bulgaria in the category „Least Concern” and in the Red book for Bulgaria in category “Rare”. Asparagus officinalis L. is included in the the Biological Diversity Act in the category „Under the protection and regulated use of nature”. The presence of Adonis vernalis L. and Jurinea ledebourii Bunge is associated with their migration from the nearby steppe region. 41 A characteristic of model habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) The anthropophytes are 65 species (73.86%). The high number is due to the artificial origin of the habitat and adjacent to farmland. The anthropogenic influence due to the presence of: 1. Improved access to the habitat by a system roads. 2. Pollution by garbage from the shepherds and farm workers. 3. Arable land in the vicinity. shrubs to trees (sh-t) with 4 species each of them (8.33%). The transition group of annual to biennial herbaceous plants (а-b) has 3 species (6.25%). The biennial herbaceous plants (b) are 2 species (4.17%). The transition group of biennual to perennial herbaceous plants (b-р) has one species (2.08%) only. The largest number of species (19) has circumboreal origin. Secondly, are species with Mediterranean origin (10). The next are species with european origin – they are 7 species. The cosmopolitan are 4 species. Three species have Pontic origin. Three of the species are adventive elements. One of the species has Oriental-Turanian origin. One of the species is Balkan subendemite – Carduus candicans Waldst. et Kit. In the habitat is meeting once Tertiary relict – Fraxinus excelsior L. Its coverage is less than 5%. The number of individuals is in the range 6 – 50. The reason for its presence can be traced in the transference of fruit from neighboring areas. One species with protection statute is established – Artemisia pedemontana Balb. It is included in IUCN Red List for Bulgaria in the category HABITAT 10 Forest shelter belt formed by Robinia pseudoacacia L. It is located as a first shelter belt east of the highway between Durankulak village and Durankulak Checkpoint, municipality Shabla. The average altitude is 20 m. The first half of the shelter belt is oriented in a west – east direction. The second half of the shelter belt is oriented in a northwest – southeast direction. The territory has no inclination. The area is 15 dka. The length of the shelter belt is 1 000 m, the width – 15 m, and the height – 7 m. The soil type is Leptosols, and the soil subtype is rendzic Leptosols. The bedrock is limestone. The cover of the tree vegetation is 60%, the cover of the shrub vegetation is 10% and the cover of the herbaceous vegetation is 80%. In the shelter belt have been indetified 48 species of vascular plants from 40 genera and 19 families. It is the most poor of plant species from the shelter belts. The families with greatest number of genera are as follows: Asteraceae – 9 (22.50%) and Рosaceae – 5 (12.50%). The families with greatest number of species are as follows: Asteraceae – 10 (20.83%), Рosaceae – 7 (14.58%) and Rosaceae – 6 (12.50%). The genera with greatest number of species are as follows: Prunus – 3 (6.25%), Artemisia, Elymus, Euphorbia, Fraxinus, Galium, Lamium and Poa – with 2 species each of them (4.17%). With the highest percentage of coverage (4) is Robinia pseudoacacia L., followed by species Elymus repens (L.) Gould. and Elymus hispidus (Opiz) Meld. (3) and Poa pratensis L. (2а). With the lowest percentage of coverage (1) are 16 species. Each of the remaining 28 species has coverage 2m. The perennial herbaceous plants (p) are most – they are 19 species (39.58%). Secondly, are annual herbaceous plants (a) with 9 species (18.75%). Thirdly, are the trees (t) with 6 species (12.50%). The next are the shrubs (sh) and the transition group of „Endangered”, in the Red book for Bulgaria in the category „Threatened with extinction” and in the Biological Diversity Act in the category „Protected”. Its presence can be explained by the transfer of the fruits by wind and finding favorable conditions, associated with good light in the shelter belt. The anthropophytes are 42 species (87.50%). Extremely high number of them due to the artificial origin of the habitat and adjacent to farmland. The anthropogenic influence due to the presence of: 1. Improved access to the habitat by a system roads. 2. Arable land in the vicinity. 4. Conclusions From natural habitats are established most taxonomical diversity in Western Pontic Paeonian steppes near to Bejanovo village, and least taxonomical diversity in Rupicolous calcareous or basophilic grasslands of the Alysso-Sedion albi between Onogur and Efreitor Bakalovo villages. From forest shelter belts are established most taxonomical diversity in Forest shelter belt formed by Gleditsia triacanthos L., and least taxonomical diversity in Forest shelter belt formed by Robinia 42 Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) pseudoacacia L. between Durankulak village and Durankulak Checkpoint. The families with greatest number of genera and species are as follows: Asteraceae, Рoaceae, Rosaceae and Lamiaceae. In the analysis of the biological types was established pattern common to all habitats: most numerous are perennial and annual herbaceous plants. This confirms the results of research of Kozuharov et all. [33]. In all habitats are most floristic elements with circumboreal, European and Mediterranean origin. The Tertiary relicts, which are established, are trees and one shrub. They often have a secondary origin for the habitats. The species with protection statute in natural habitats are most in Western Pontic Paeonian steppes near to Bejanovo village – they are 8 species. In the other habitats that number varies from 2 to 4 species. In the forest shelter belts can be found a small number of species with protection statute. Most often they have gone from adjacent areas. The number of anthropophytes in the natural habitats is a significant – from 60.13% to 77.67%. [5] NATURA 2000 Standard Data Form for Protected Area „The Valley of Batova River” (BG0000102), Ministry of Environment and Waters of Bulgaria, 15 pp. [6] NATURA 2000 Standard Data Form for Protected Area „Kraimorska Dobrudja” (BG0000130), Ministry of Environment and Waters of Bulgaria, 19 pp. [7] NATURA 2000 Standard Data Form for Protected Area „Durankulak Lake” (BG0000154), Ministry of Environment and Waters of Bulgaria, 13 pp. [8] NATURA 2000 Standard Data Form for Protected Area „Shabla – Ezeretz Lake” (BG0000156), Ministry of Environment and Waters of Bulgaria, 16 pp. [9] NATURA 2000 Standard Data Form for Protected Area „Suha reka” (BG0002048), Ministry of Environment and Waters of Bulgaria, 13 pp. [10] NATURA 2000 Standard Data Form for Protected Area „Kardam” (BG0000569), Ministry of Environment and Waters of Bulgaria, 10. [11] NATURA 2000 Standard Data Form for Protected Area „Izvorovo – Kraishte” The reasons about this are mainly the following: the strong fragmentation of natural habitats, arable land in the vicinity – like source of anthropophytes, improved access to the habitats and their accessibility for people and domestic animals. In the forest shelter belts the anthropophytes quite naturally are more – from 73.86% to 87.50%. (BG0000570), Ministry of Environment and Waters of Bulgaria, 10 pp. [12] NATURA 2000 Standard Data Form for Protected Area „Rositza – Loznitza” (BG0000572), Ministry of Environment and Waters of Bulgaria, 12 pp. [13] NATURA 2000 Standard Data Form for Protected Area „Complex „Kaliakra” (BG0000573), Ministry of Environment and Waters of Bulgaria, 27 pp. [14] TZONEV, R., Rusakova, V., Dimitrov, М. Dimova, D., Belev, T. Kavrakova, V., 2004. Proposals for habitats for inclusion to Annex I on Council Directive 92/43/EEC of the European Community to protect natural habitats and of wild fauna and flora and Interpretation handbook of habitats in the European Union EUR 15/2, Report, World Wildlife Fund, Danube – Carpathian Program (WWF, DCP). [15] KAVRAKOVA, V., Dimova, D., Dimitrov, М., Tzonev, R., Belev, T. (editors), 2005. A guidance for identifying the habitats of European importance in Bulgaria, Geosoft, Sofia, 128. 5. References [1] VELEV, S., 2002. Climatic zoning, in Kopralev, I. (main ed.). Geography of Bulgaria. Physical and socio-economic geography, Institute of Geography, BAS, Farkom, Sofia, 760 pp. [2] NINOV, N., 2002. Soils, in Kopralev, I. (main ed.). Geography of Bulgaria. Physical and socioeconomic geography, Institute of Geography, BAS, Farkom, Sofia, 760 pp. [3] KITANOV, B., Penev, I., 1980. Flora of Dobrudja, Nauka i Izkustvo, Sofia, 630 pp. [4] BONDEV, I., 1991. The vegetation of Bulgaria. Map in М 1:600 000 with explanatory text, University Press St. Kliment Ohridski, Sofia, 183 pp. 43 A characteristic of model habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) [16] KOZUHAROV, S. (ed.), 1992. Identifier of the vascular plants in Bulgatia, Nauka i izkustvo, Sofia, 788 pp. [17] Flora of PR Bulgaria, Vol. І-Х, 1963-1995, Publishing House of BAS, Sofia. [18] CHASE, M. (corresponding author), 2003. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II, The Linnean Society of London, Botanical Journal of the Linnean Society, 141: 399–436. [19] PETROVA, А., Anchev, М. Palamarev, Е., 1999. How to recognize the plants in our nature. Char identifier. Prosveta, Sofia, 837 pp. [20] WESTHOFF, V., Maarel, E., 1973. The BraunBlanquet Approach in: Tuxen, R. (Ed.), Handbook of vegetation science. Dr. W. Junk b. v. Publishers the Hague, p. 619-704. [21] ASIOV B., Petrova A., Dimitrov D., Vasilev R., 2006. Conspectus of the Bulgarian vascular flora. Distribution maps and floristic elements, Bulgarian Biodiversity Foundation, Sofia, 452 p. [22] GRUEV, B., Kuzmanov B., 1994. General biogeography, University Press St. Kliment Ohridski, Sofia, 498 pp. [28] Convention on International Trade in Endangered Species of Wild Fauna and Flora, State Gazette number 6 from 21 Januari 1992. [29] Red book of PR Bulgaria, Vol. 1, Plants, 1984, Publishing House of BAS, Sofia, 447 pp. [30] PETROVA А., Vladimirov V. (eds.), 2009. Red List of Bulgarian vascular plants, Phytologia Balcanica 15 (1): 63–94. [31] Order number RD-72 from 3 februari 2006 for special arrangements for the conservation and use of medicinal plants, State Gazette number 16 from 21 Februari 2006. [32] STEFANOV, B., Kitanov B., 1962. Kultigenen plants and kultigenen vegetation in Bulgaria, Publishing House of BAS, Sofia, 275 pp. [33] KOZUHAROV, S., Dimitrov, D., Lazarova, М., Kozuharova, Е., 1997. A characteristic of the flora and the vegetation of the natural plant complexes in Southern Dobrudja, Conference proceedings „Dobrudja and Kaliakra”, Academic publishing of Higher Agricultural Institute, Plovdiv, p. 42-58. [23] PEEV, D., 2001. National park Rila. Management plan 2001 – 2010. Adopted by Resolution №522 of Council of Ministers on 04.07.2001, Sofia, 338 pp. [24] BOŽA, P., Anačkov G., Igić R., Vukov D., Polić D., 2005. Flora “Rimskog šanca” (Vojvodina, Srbija), 8th Symposium on the flora of Southeastern Serbia and Neighbouring Regions, Niš, 20-24.06.2005, Abstracts, рр. 55. [25] PEEV, D., Kozuharov S., Anchev M., Petrova A., Ivanova D., Tzoneva S., 1998. Biodiversity of Vascular Plants in Bulgaria, In: Curt Meine (ed.), Bulgaria's Biological Diversity: Conservation Status and Needs Assessment, Volumes I and II, Washington, D.C., Biodiversity Support Program, pp. 55–88. [26] Council Directive 92/43/EEC of the European Community to protect natural habitats and of wild fauna and flora. [27] Biological Diversity Act, State Gazette number 77 from 9 august 2002, pp. 9–42. Amended in State Gazette number 94 from 16.11.2007. 44 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 FLORISTIC ASPECTS OF THE HILLS OF CAMENA VILLAGE (TULCEA COUNTY) Marius FĂGĂRAŞ Ovidius University of Constanţa, Faculty of Natural and Agricultural Sciences, Department of Biology and Ecology, Mamaia Blvd, No. 124, 900527, Constanţa, Romania, fagarasm@yahoo.com __________________________________________________________________________________________ Abstract: This paper presents the flora on the hills in the vicinity of Camena locality. These hills have volcanic origin and are located in the south-east of the Babadag plateau. The hilly landscape with spectacular rock formations, the substrate made up of acidic volcanites and the climate specific to the forest steppe are the main factors that determined the variety of the vegetation made up of steppe meadows, rock formations, forests and bushes. The area is characterized by the presence of a considerable number of floral rarities, some endemic, other rare, vulnerable or endangered at national level. Despite all these, the flora of the area is still little known as there are no specialized papers. The enumeration of the vascular flora is accompanied by an analysis of the biological forms, of the floral elements, of the ecological categories, but also of the floral rarities present on these hills. Keywords: Camena hills, flora, life forms, floristic elements, ecological categories, rare and threatened flora. ___________________________________________________________________________ 1. Introduction The hills of Camena are located south of Ciucurovei Hills, in the south-east of the Babadag Plateau, in the vicinity of Camena village (Tulcea County). They are volcanic hills (Fig. 7), with a maximum altitude of approx. 190 meters, located at the southern end of the Peceneaga-Camena crevice which separates the Northern Dobrogea Plateau from the Central Dobrogea Plateau. The Hills of Camena look like a wide saddle framed towards the north-west and south-east by the hydrographic basins of two valleys: Camena valley and Ciamurlia valley. In the southern part of these hills is the Altan Tepe copper pyrite mine. The geological layer is made up of rhyolites (quartz porphyry) of Paleozoic age, volcanic rocks (acid volcanites) colored in pink-red, reddish-brown and violet. In the plane zone and on the eroded inclines, the rhyolites emerge on the surface as spectacular rock formations. Towards the base of the hills, the rocks are covered by a layer of loess (3-4 meters thick). The soils are represented by chernozem and lithosoils, the latter being present especially in the rocky zones. ISSN-1453-1267 The climate is temperate-continental, with average annual temperatures of 10.5-110 C, while the average annual precipitations range between 450 and 500 mm/year. As vegetation type, the Hills of Camena fit within the forest steppe zone. The vegetation is made up of steppe meadows, rock vegetation (on the plateaus), thermophile forests and bushes. The Hills of Camena represent an area of the Babadag Plateau which is interesting from the geological, landscape and botanical point of view, firstly because of the volcanic origin of the hills, of the rhyolites disposed as spectacular rock formations and of the floral rarities which can be encountered in this area. Despite these, the flora of these hills is little known and limited to the quotation of species in older specialized literature [1, 2, 3, 4, 5]. 2. Material and Methods The field researches have been done between years 2008-2010, during the entire vegetation season in order to cover all the phenology stages. The plant taxa nomenclature follows the Flora ilustrată a României. Pteridophyta et © 2010 Ovidius University Press Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) Spermatophyta [6], Flora Europaea [7, 8] and Flora României [4]. The life forms, floristic elements and ecological categories have been established on the base of the synthesis works Conspectul florei cormofitelor spontane din România [9] and Flora ilustrată a României. Pteridophyta et Spermatophyta [6]. The rare and threatened plant species was done according to the Romanian Red List [10] and the Romanian Red Book of the vascular plants [11]. hemicryptophytes (42.13%) and the annual and biennial terophytes (35.95%), present especially in the steppe meadows. Poorly represented are the phanerophytes (9.55%), which are included in forests (with Quercus petraea subsp. dalechampii, Quercus pubescens, Carpinus orientalis, Fraxinus ornus, Prunus mahaleb, Tilia tomentosa) and bushes (with Crataegus monogyna, Prunus spinosa, Cotinus coggygria, Ligustrum vulgarae, Rosa canina, Cornus mas) in the investigated area. The category of phanerophytes also includes alien species encountered in these hills, some of them invasive or potentially invasive (Robinia pseudacacia, Ailanthus altissima, Elaeagnus angustifolia). The geophytes (7.30%) and the camephytes (5.05%) are perennial species found especially in the grassy blanket from forests or forest edges. 3. Results and Discussions The floristic researches carried out on the hills of Camena village have lead to identification of 178 vascular taxa (168 species and 10 subspecies) (Table 1). Taxa found in the studied area belong to 46 families and 38 classes of Spermatophyta Divisio. The following families are well represented as number of taxa (Fig. 1): Asteraceae (12,35%), Lamiaceae (10,67%), Poaceae (9,55%), Rosaceae si Brassicaceae (5,05%), Liliaceae, Caryophyllaceae si Fabaceae (cate 4,49%), Apiaceae (3,93%), Boraginaceae (3,37%), Ranunculaceae (2,80), Geraniaceae (2,24) and Scrophulariaceae (1,68%). G 7,30% PH 9,55% CH 5,05% H 42,13% TH 35,95% 14 12 Fig. 2. The spectrum of biological forms (H-hemicriptofite; TH-therofite; PH-fanerofite; G-geofite; CH-camefite) 10 8 % 6 4 Among the floristic elements (Table 2 and Figure 3), the dominant species are the Eurasian (35.39%) and Pontic (26.40%) ones, followed at great distance by other categories of geoelements: European (8.98%), Central-European (6.17%), Mediterranean and sub-Mediterranean (6.17%), Balkan (5.61%), circumpolar (1.68%), AtlanticMediterranean, Taurean-Balkan, Carpatho-BalkanCaucasian, and endemic (each with 0.56%). The large proportion of Pontic species reflects on the one side the dominance of the steppe meadows in the studied area, and on the other side, the nearness of the Razelm-Sinoe lagoon complex (located approx. 10 km east), which belongs to the Pontic biogeographic region. Among the categories 2 0 AST LAM POA ROS BRAS LIL CARY FAB API BOR RAN GER SCR families Fig. 1. Most important botanical families as the number of species (AST-Asteraceae; POAPoaceae; LAM-Lamiaceae, ROS-Rosaceae; LILLiliaceae;CARY-Caryophyllaceae; FAB-Fabaceae; API-Apiaceae; BRAS-Brassicaceae; RAN-Ranunculaceae; BOR-Boraginaceae; GER-Geraniaceae; SCR-Scrophulariaceae) From the point of view of the biological forms (Fig.2), the dominant ones are the 46 Marius Făgăraş / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) of Pontic elements (Fig. 4), the best represented in the studied area are: the Pontic-Mediterranean (55.31%), Pontic-Balkan (17.02%), Pontic proper (10.63%), Pontic-Pannonian-Balkan (8.51%), Pontic-Pannonian (4.25%), Pontic-Caucasian and Pontic-Central-European (2.12% each). The arid climate in the area of these hills is favorable for the large number of species of southern origin (Mediterranean, Sub-Mediterranean, Balkan), that make up a percentage of 11.78%. Table 2. The percentages of floristic elements in the studied area Floristic elements Eua Eur Euc Pont Med + subMed Balc Subcategories No. Eua Eua(Cont) Eua(Med) Eur Eur(Cont) Eur(Med) SE Eur Euc Euc -Med EucsubMed Euc-Balc Pont Pont-Med Pont-Balc Pont-Pan Pont-PanBalc Pont-Cauc Pont-Euc Med subMed 25 17 21 7 3 4 2 4 5 1 Balc Balc-Pan Balc-Anat Balc-Cauc Balc-PontAnat 4 2 1 2 1 1 5 26 8 2 4 1 1 10 1 TaurBalc CarpBalcCauc Atl-Med - 1 0,56 - 1 0,56 - 1 0,56 Circ End - 3 1 1,68 0,56 Balc 5,61% % Circ Adv 2,24% 1,68% Cosm 5,05% Others 2,37% Eua 35,39% Med 6,17% Pont 26,40% 35,39 Euc 6,17% Eur 8,98% 8,98 Fig. 3. The spectrum of floristic elements 6,17 Pont-Pan-Balc 8,51% Pont-Cauc 2,12% Pont-Euc 2,12% Pont 10,63% Pont-Balc 17,02% Pont-Pan 4,25% 26,40 Pont-Med 55,31% Fig. 4. The spectrum of the Pontic elements Among the ecological categories connected to soil humidity (Fig. 5), we can remark the considerable percentage of xero-mesophile (57.3%) and xerophile (24.71%) species, components of the steppe meadows and of rock formation vegetation. The mesophile species (14.6%) are present especially in the forested area of the hills. The eurythermal species have a smaller percentage (2.8%). 6,17 5,61 47 Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) From the point of view of the preference for temperature (Fig. 5), the micro-mesothermal (46.62%) and moderately thermophile (37.64%) species have considerable percentages, as they are regular in the silvosteppe zone which is made up of steppe meadows and forests. The thermophile species (6.17%) are encountered either in the steppe meadows or in the rock formations. From the point of view of the preference for soil pH (Fig. 5), the higher percentages are held by poor acid-neutrophile (53.37%), acidic-neutrophile (17.97%) and euryionic species (20.22%). We must remark the high percentage of acidophile species (2.8%), grouped on the acidic volcanites that make up the rock formations in the plateau zone. Table 3. The rare and threatened taxa in the Camena Hills area No Name of the taxa Floris tic eleme nts IUCN categories [11] IUCN categories [10] 1. Achillea coarctata R - 2. Allium flavum subsp. tauricum Campanula romanica Crocus reticulatus PontBalc TaurBalc End R - V/R EN PontMed Balc V - V/R VU PontPanBalc Balc R - R VU PontBalc Med R LR R - Pont R VU PontBalc R - Balc V/R - PontMed SE Eur Pont PontMed PontCauc Pont Balc E/R - R - R R EN R VU R R - 3. 4. 5. 70 6. U% 60 T% 50 Dianthus nardiformis Echinops ritro subsp. ruthenicus R% 40 7. % 30 8. 20 Galanthus plicatus Iris sintenisii 10 9 0 1-1,5 2-2,5 3-3,5 4-4,5 5-5,5 6 0 ecological categories 10 . 11 Fig. 5. The spectrum of ecological categories The 19 rare and endangered taxa (Table 3) represent 10.67% of the total species and subspecies identified in the Hills of Camena. A more important element is the presence of the endemic species Campanula romanica in the area, but also of other rare and very rare plants at national level, mentioned in the Red Book of vascular plants of Romania [11]: Dianthus nardiformis (Fig. 8), Silene compacta, Moehringia jankaea, Iris sintenisii, Salvia aethiopis, Sempervivum zeleborii, Galanthus plicatus, Nectaroscordium siculum subsp. bulgaricum, Achillea coarctata, Crocus reticulatus, etc. In terms of the main endangered categories (Fig. 6), 1 taxon (0.56%) is endangered, 4 taxa (2.24%) are vulnerable, while 14 other (7.86%) are rare, with small populations at national level. 12 13 14 48 Myrrhoides nodosa Moehringia jankae Nectaroscordium siculum subsp. bulgaricum Paeonia peregrina Salvia aethiopis 15 16 Sempervivum zeleborii Seseli campestre Silene compacta 17 Stipa ucrainica 18 19 Syrenia cana Thymus zygioides Marius Făgăraş / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) E 0,56% V 2,24% 5. References R 7,86% [1] PRODAN I., 1934-Conspectul florei Dobrogei I, Bul. Acad. de Înalte St. Agronomice, Tipogr. Naţională S.A., Cluj, 5, 1. [2] PRODAN I., 1935-1936 - Conspectul florei Dobrogei II, Bul. Acad. de Înalte St. Agronomice, Tipogr. Naţională S.A., Cluj, 6. [3] PRODAN I., 1938 - Conspectul florei Dobrogei III, Bul.Facult. de Agronomie, Cluj, Tipogr. Cartea Românească., 7. [4] SĂVULESCU T. (ed.), 1952-1976 - Flora României, vol. I-XIII, Edit.Academiei Române, Bucureşti. [5] DIHORU GH., DONIŢĂ N., 1970 - Flora şi vegetaţia podişului Babadag, Edit. Academiei R.S.R., Bucureşti. [6] CIOCÂRLAN V., 2000 - Flora ilustrată a României (Pteridophyta et Spermatophyta), Edit. Ceres, Bucureşti. [7] TUTIN T.G. HEYWOOD V.H., BURGES N.A., MOORE D.M., VALENTINE D.H., WALTERS S.M. & WEBB D.A. (eds), 19641980 - Flora Europaea, Vols. 1-5, Cambridge, Cambridge University Press. [8] TUTIN T.G. HEYWOOD V.H., BURGES N.A., MOORE D.M., VALENTINE D.H., WALTERS S.M. & WEBB D.A. (eds., assist. by AKEROYD J.R & NEWTON M.E.; appendices ed. by MILL R.R.), 1993 (reprinted 1996) - Flora Europaea, 2nd ed., Vol. 1, Cambridge, Cambridge University Press. [9] POPESCU A., SANDA V., 1998 - Conspectul florei cormofitelor spontane din România, Acta Botanica Horti Bucurestiensis, Edit. Universităţii din Bucureşti. [10] OLTEAN M., NEGREAN G., POPESCU A., ROMAN N., DIHORU GH., SANDA V., MIHĂILESCU S., 1994 - Lista roşie a plantelor superioare din România, Studii, Sinteze, Documente de Ecologie, Bucureşti, (1): 1-52. [11] DIHORU GH., NEGREAN G., 2009 - Cartea Roşie a plantelor vasculare din România, Edit. Academiei Române, Bucureşti. NT 89,33% Fig. 6. The spectrum of sozological categories 4. Conclusions The research realized between 2008 and 2010 led to the identification of 178 vascular taxa which, from the taxonomical point of view, belong to 46 families and 38 orders. From the point of view of the biological forms, the dominant are the hemicryptophytes and terophytes, components of the steppe meadows in the area of Camenei Hills. The phanerophytes, camephytes and geophytes are present especially in the forested areas of these hills. Alongside the Eurasian species, well represented in the studied area are also the Pontic elements specific to westPontic steppes, but also those of southern origin (Mediterranean, sub-Mediterranean and Balkan), an expression of a climate with sub-Mediterranean nuances. Among the ecological categories of plants established according to their preference for substrate humidity, air temperature and soil pH, the predominant species are xero-mesophile, micromesothermal and moderately-thermophile ones, as well as the poorly acid-neutrophile ones. Of the total identified taxa, the rare and endangered species represent 10.67%. The important local populations of certain endemic and rare species at national level place the Camena Hills in the northern Dobrogea zones important from the conservation point of view. 49 Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) Fig. 7. General aspect of volcanic hills of Camena Fig. 8. Dianthus nardiformis on the volcanic rocks of Camena 50 Marius Făgăraş / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) Table 1. The list of the vascular plants of Camena Hills No. Taxa Family 1. 2. 3. 4. Achillea coarctata Achillea setacea Adonis flammaea Agropyron cristatum subsp. pectinatum Agropyron ponticum Ailanthus altissima Ajuga chamaepytis subsp. ciliata Ajuga genevensis Alliaria petiolata Allium flavum subsp. tauricum Allium rotundum Alyssum alyssoides Anagalis arvensis Androsace maxima Anemone sylvestris Anthemis ruthenica Anthriscus cerefolium subsp. trichosperma Artemisia absinthium Artemisia austriaca Asparagus verticillatus Asperula cynanchica Asperula tenella Ballota nigra Bassia prostrata Berteroa incana Brachypodium sylvaticum Bromus hordeaceus Bromus sterilis Bromus tectorum Buglossoides arvensis Buglossoides purpurocaerulea Calepina irregularis Camelina microcarpa Campanula romanica Campanula sibirica Cardaria draba Carduus acanthoides Carpinus orientalis Carthamus lanatus Centaurea cyanus Centaurea diffusa 5. 6. 7. 8. 9. 10. 11. 12 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. AST AST RAN POA Life forms H H TH H Floristic elements Pont-Balc Eua(cont) Pont-Med Pont-Euc Ecological categories U1,5 T4,5 R4,5 U2 T3 R5 U2 T3,5 R3,5 U2 T4 R4,5 POA SIM LAM LAM LIL LIL LIL BRAS PRIM PRIM RAN AST API H(G) PH TH H TH-H G G TH TH TH H TH TH Pont-Balc Adv Pont-Med Eua(cont) Eua Taur-Balc Euc(Med) Eua(Cont) Cosm Eua(Cont) Eua(cont) Eur(Cont) Pont-Med U1,5 T4,5 R4,5 U0 T0 R0 U2,5 T4 R3 U2 T3 R4 U3 T3 R4 U1,5 T4 R4 U2 T4 R4 U1 T3 R0 U3 T3,5 R0 U2 T4 R4 U2 T3,5 R4 U2 T4 R4 U2,5 T4 R4 AST AST LIL RUB RUB LAM CHEN BRAS POA POA POA POA BOR BOR BRAS BRAS CAMP CAMP BRAS AST CORY AST AST AST H(CH) CH G H H H CH TH H TH TH TH TH H-G TH TH H H H TH PH TH TH TH Eua Eua(cont) Pont-Balc Pont-Med Pont-Balc Euc Eua(cont) Eua(cont) Eua(Med) Eua Eua(Med) Eua(cont) Eua Euc-subMed Pont-Med Eua End Eua(cont) Eua(Med) Eur(Med) Balc-Cauc Pont-Med Med(Cosm) Pont-Balc U2 T3 R4 U2 T4 R4,5 U1 T4,5 R4 U2 T3 R5 U2 T4 R4 U2 T3,5 R4 U1,5 T4 R4,5 U2 T3 R4 U3 T3 R4 U0 T3 R0 U2 T4 R4 U1,5 T3,5 R0 U0 T0 R4 U2,5 T4 R4,5 U2 T4 R3 U3 T3 R0 U1,5 T4 R0 U2,5 T4 R4 U2 T4 R4 U2 T3 R0 U3 T4 R4,5 U2,5 T4 R0 U3 T4 R0 U2 T4 R0 51 Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. Cerastium brachypetalum Chamomilla recutita Chondrilla juncea Chrysopogon gryllus Cichorium intybus Conium maculatum Convolvulus arvensis Convolvulus cantabricus Conyza canadensis Cornus mas Coronilla varia Corydalis cava Cotinus coggygria Crataegus monogyna Crepis sancta Crocus reticulatus Crupina vulgaris Cynanchum acutum Cynodon dactylon Daucus carota Dianthus nardiformis Dichanthium ischaemum CARY AST AST POA AST API CONV CONV AST CORN FAB FUM ANAC ROS AST IRID AST ASCL POA API CARY POA TH TH H H H TH-TH H(G) H TH PH H G PH PH TH G TH H G(H) TH CH H Med Eua(Med) Eua Med Eua Eua Cosm Pont-Med Adv(Am.N) Pont-Med Eua(Med) Euc Pont-Med Eur Pont-Balc Pont-Med Pont-Med Pont-Med Cosm Eua(Med) Balc Eua(Med) U3 T3 R0 U2,5 T3,5 R5 U1,5 T3,5 R4 U1,5 T4 R4 U3 T0 R3 U3 T3 R3 U2,5 T3,5 R3,5 U1,5 T3,5 R4 U2,5 T0 R0 U2 T3,5 R4 U2 T3 R4 U3 T3 R0 U2 T4,5 R4 U2,5 T3,5 R3 U1,5 T4 R4 U2,5 T4 R3 U2 T3,5 R0 U2,5 T4 R0 U2 T3,5 R0 U2,5 T3 R0 U1,5 T4,5 R4,5 U1,5 T5 R3 64. 65. 66. 67. 68. 69. 70. 71. Echinops ritro subsp. ruthenicus Elaeagnus angustifolia Elymus repens Erodium cicutarium Eryngium campestre Erysimum diffusum Euphorbia agraria Euphorbia nicaeensis AST ELEG POA GER API BRAS EUPH EUPH H PH G TH H H H H U1,5 T4 R4,5 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. Festuca valesiaca Filipendula vulgaris Fragaria viridis Fraxinus ornus Fumaria rostellata Galanthus plicatus Galium humifusum Geranium divaricatum Geranium pussilum Geranium rotundifolium Geum urbanum Glechoma hirsuta Hieracium bauhinii Hieracium pilosella Holosteum umbellatum Hypericum perforatum Iris sintenisii POA ROS ROS OLE FUM AMAR RUB GER GER GER ROS LAM AST AST CARY HYP IRID H H H PH TH G H TH TH TH H H(CH) H H TH H G Pont-Pan-Balc Adv Circ Cosm Pont-Med Eua(Cont) Pont-Med Pont-Pan-BalcAnat Eua(cont) Eua Eur(Cont) Med Euc-Balc Taur-Cauc Pont-Balc Eua(Med) Eur(Med) subMed Eua(Med) Pont-Med-Euc Euc Eua Eua(Med) Eua Pont-Balc 52 U0 T0 R0 U2,5 T0 R0 U1 T5 R4 U1,5 T3 R4 U2 T4 R0 U1,5 T5 R5 U1 T5 R4 U2,5 T3 R4,5 U2 T4 R3 U1,5 T3,5 R5 U3 T0 R3,5 U3 T4 R3 U2 T4 R4,5 U2,5 T3 R4 U2,5 T3 R0 U2 T3,5 R4 U3 T3 R4 U2,5 T3 R4 U1,5 T3 R3,5 U2 T0 R2 U2 T3,5 R0 U3 T3 R0 U2 T4 R4 Marius Făgăraş / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. Koeleria macrantha Lamium amplexicaule Lappula barbata Lathyrus tuberosus Ligustrum vulgare Linum austriacum Malva sylvestris Marrubium peregrinum Marrubium vulgarae Medicago lupulina Medicago minima Melica ciliata Melilotus alba Minuartia setacea Moehringia jankae Myosotis stricta Myrrhoides nodosa Nectaroscordum siculum subsp. bulgaricum Nigella arvensis Nonea atra Onopordum acanthium Onosma visianii Origanum vulgare Orlaya grandiflora Ornithogalum refractum Paeonia peregrina Papaver dubium Papaver rhoeas Petrorhagia prolifera Phleum phleoides Phlomis tuberosa Pinus nigra Plantago lanceolata Poa angustifolia Polycnemum majus Polygonum aviculare Polygonatum latifolium Potentilla argentea Potentilla recta s.l. Prunus mahaleb Prunus spinosa Quercus petraea subsp. dalechampii Quercus pubescens Ranunculus oxyspermus Reseda lutea Robinia pseudacacia POA LAM BOR FAB OLE LIN MALV LAM LAM FAB FAB POA FAB CARY CARY BOR API LIL H TH TH-TH H(G) PH H TH(H) H H(CH) TH(H) TH H TH CH H TH TH G Circ Eua(Med) Pont-Med Eua(Med) Eua(Med) Eua(cont) Eua(Cosm) Eua(Med) Eua(Med) Eua Eua(Med) Eur(Med) Eua Pont Pont Eua(Med) Med Pont-Balc U2 T4 R5 U2,5 T3,5 R0 U2 T3,5 R4 U2 T4 R4 U2,5 T3 R3 U1,5 T3,5 R4 U3 T3 R3 U2 T4 R0 U1 T4 R4 U2,5 T3 R4 U1,5 T4 R4 U1,5 T4 R4 U2,5 T3 R0 U1,5 T0 R4 U1 T4 R4,5 U2 T0 R2,5 U2,5 T4,5 R4,5 U3,5 T3,5 R3,5 RAN BOR AST BOR LAM API LIL PAE PAP PAP CARY POA LAM PIN PLAN POA CHEN POLG LIL ROS ROS ROS ROS FAG TH TH TH H H TH G H(G) TH TH TH H H PH H H TH TH G H H PH PH PH U2 T4 R4 U2 T4 R3 U2,5 T4 R4 U1,5 T4,5 R4,5 U2 T3 R3 U2 T3,5 R4 U2 T3,5 R4 U2 T3,5 R5 U2 T3,5 R3 U3 T3,5 R4 U1,5 T4 R3 U2 T3 R4 U2,5 T3,5 R4 U0 T0 R0 U3 T0 R0 U2 T3 R0 U1,5 T4,5 R4 U2,5 T0 R3 U3 T3,5 R4 U2 T4 R2 U1,5 T3,5 R4 U2 T3 R4,5 U2 T3 R3 U2,5 T2,5 R0 FAG RAN RES FAB PH H TH(H) PH Pont-Med Balc-Anat Eua(Med) Pont-Pan-Balc Med Euc-Med Balc-Pan-Cauc Balc Eur Cosm Pont-Med Eua(cont) Eua(Cont) Eua Eua Eua Eua Cosm Pont-Pan-Balc Eua Eur(Cont) Med Eur(Med) E.Med.-CarpBalc Med Balc-Cauc Eua(Med) Adv(Am.N) 53 U1,5 T5 R5 U2,5 T3 R3 U2 T3 R0 U2,5 T4 R0 Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. Rosa canina Rumes acetosella subsp. acetoselloides Salvia aethiopis Salvia nemorosa Salvia nutans Sambucus nigra Saxifraga tridactylites Scilla bifolia Scleranthus annuus Scleranthus perennis Sedum maximum Sempervivum zeleborii Senecio jacobaea Seseli campestre Sideritis montana Silene compacta Sisymbrium orientale Solidago virgaurea Sonchus oleraceus Stachys germanica Stachys recta Stipa capillata Stipa ucrainica Syrenia cana Teucrium chamaedrys Teucrium polium subsp. capitatum Thalictrum minus Thlaspi perfoliatum Thymus pannonicus Thymus zygioides Tilia tomentosa Tragopogon dubius Trifolium arvensae Trifolium campestre Trifolium echinatum Urtica dioica Valerianella lasiocarpa Verbascum phlomoides Veronica austriaca subsp. jacquinii Veronica teucrium Vinca herbacea Vincetoxicum hirundinaria Viola kitaibeliana Viola odorata ROS POLG PH H Eur SE Eur U2 T3 R3 U2 T3 R2 LAM LAM LAM CAPR SAX LIL CARY CARY CRAS CRAS AST API LAM CARY BRAS AST AST LAM LAM POA POA BRA LAM LAM H H H PH TH G TH-TH H(CH) H CH H H TH TH TH H TH H H H H TH CH CH Pont-Med Pont-Med Pont-Pan Eur Eua Euc Eua Eur Eur SE Eur Eua Pont Eua Pont-Med Pont-Med Circ Cosm Pont-Med Pont-Med-Euc Eua(Cont) Pont-Cauc Pont Euc(Med) Med U2 T5 R0 U2 T4 R4 U1 T5 R5 U3 T3 R3 U2 T3,5 R4 U3,5 T3 R4 U2 T3 R2 U3 T0 R3 U2,5 T0 R4 U1,5 T3,5 R4,5 U2,5 T3 R3 U2,5 T4 R4 U2 T4 R4 U2 T4 R4 U2,5 T4 R3 U2,5 T3 R3 U3 T0 R0 U2 T4 R3 U2 T5 R5 U1 T5 R4 U1 T4 R4 U1,5 T4 R4 U2 T4 R4 U1,5 T4 R4,5 RAN BRAS LAM LAM TIL AST FAB FAB FAB URT VAL SCR SCR H TH CH CH PH TH TH TH TH H TH TH H Eua Eua Pont-Pan Balc Balc-Pan Euc(Med) Eua(Med) Eur Med Cosm Balc-Pont-Anat Euc(Med) Pont-Med-Euc U2 T4 R4 U2,5 T3,5 R4,5 U1,5 T3,5 R4 U1,5 T4 R4,5 U2,5 T3,5 R3 U2,5 T3,5 R0 U1,5 T3 R4 U3 T3 R0 U1,5 T4,5 R4 U3 T3 R4 U1,5 T5 R4 U2,5 T3,5 R4 U2 T4 R4 SCR APOC ASCL VIO VIO H H H TH H Eua(Med) Pont Eua(Cont) Pont-Med Atl-Med U1,5 T4 R4,5 U2 T5 R4 U2 T4 R4 U2 T4 R4,5 U2,5 T3,5 R4 54 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 IDENTIFICATION OF SOME ROSE GENITORS WITH RESISTANCE TO THE PATHOGENS AGENTS ATTACK *Marioara TRANDAFIRESCU, Corina GAVĂT, Iulian TRANDAFIRESCU and Elena DOROFTEI *Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology Mamaia Avenue, No. 124, Constanţa, 900552, Romania, e-mail: mtrandafirescu@yahoo.com ________________________________________________________________________________________ Abstract: In the South-Eastern Romania, as in all country the rose culture is higly praised for its ornamental value both in parks and domestic garden. In this zone of our country the rose culture is more important because the Black Seaside provide a better enviroment (82 km). Beside the growing of forign cultivars from Europe Companies, Romania has done a breeding work to develop autochtonous cultivars (Rusticana, Ambasador, Bordura de nea, Rosagold, Simina, etc.) better adapted to our local conditions. In rose breeding besides the ornamental value of these flowers (nice leaves, colours and shapes) the disease resistance has been taken into acount. Among the specific pathogens very harmful for the rose culture, one can mention: Sphaerotheca pannosa (Wallr) Lev var rosae Woron (powdery mildew), Diplocarpon rosae Wolf (black spott), Phragmidium mucronatum (Pers) Schlecht (rose rust) and Botrytis cinerea Pers (grey mold). One of the most effective methods to prevent these pathogens attack is breeding new cultivar and genically resistant genitors. These paper present the behaviour of 50 genotypes from rose collection of Fruit Growing Development of Fruit Tree Constanta and their response of such pathogens. The conditions of natural infections allowed grouping the biological material in 4 classes of resistance. This clasifications was done acording to levels of frequency (F%) and intensity (I). The rose cultivars with genetic resistance to this pathogens are: Queen Elisabeth, Foc de tabara, Rubin, Parfum, Emerald d’or, Bel Ange, Apogee. Keywords: black spott, powdery mildew, rose rust, genitors, resistance __________________________________________________________________________________________ 1. Introduction From the oldest times the rose was considered "Queen of The Flowers" due its beauty, perfume, richness in colors and multiple shapes of the grown cultivars. Therefore, the rose place is in the front of the ornamental species used for park and gardens decoration and for cut-flowers production. Unfortunately, the rose as many other cultivated plants can suffer due to the attack of some very damaging pathogens. Under favorable conditions, the pathogens can determine the partial or total defoliation of the plants, they become weak and the cut-flowers production can be diminished by quantity and quality as well. In order to prevent and control the pathogens specific to the roses, the studies carried out in Romania and in the World, were focused on identification of the species responsible for the ISSN-1453-1267 diseases occurrence and knowledge of their biology (Bedian, 1980, Bernardis, 2004, Ostaciuc, 1982, Sandu 2004, Szekelly, 1981, Wagner, 2002), and on the other hand, was investigated the efficacy of some fungicides in order to control them(Bon and coll, 1978, Morrison, 1978, Hagan and coll 1988, Losing, 1988, Qvarnstrom, 1989, Raabe, 1989, Rolim and col, 1990, etc). The results obtained in the World (Saunders, 1970; Klimenko, 1973; Simonyan, 1973; Semina, 1980, 1984; Palmer, 1978; Costlediene, 1981) and in our country (Costache, 1993, Argatu, 1993, Sekely, 1981, Wagner, 2002) clearly emphasized that the most efficient method to prevent the attack of the pathogens is the creation and extension in the culture of some roses cultivars genetically resistant to diseases. Therefore, the researches carried out during 2008-2009 at Research Station for Fruit Growing © 2010 Ovidius University Press Identification of some rose genitors with resistance... / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010) Constanta had as central objective the evaluation of some roses cultivars behavior to some key pathogens in order to identify some resistance donors genitors for further breeding works. In the reference area the pathogens of economic importance for the rose culture are Diplocarpon rosae Wolf, Sphaerotheca pannosa (Wallr)Lev var rosae Woron şi Phragmidium mucronatum (Pers) Schlecht. 10-18 mm in diameter, highly visible on the superior face of the leaves. (Fig.1). Fig. 1. Pătarea neagră a frunzelor de trandafir – Diplocarpon rosae 2. Material and Methods Under such condition, presented in table 1, the (natural) genetic resistance to the pathogen attack manifested the cultivars: Emerande d’or, Grad Premiere, Traviata, Apogee, Luchian, Monica, Coup de Foudre and Tour Eiffel. The cultivars: Baccara, Pascali, Bel Ange, Creole, Dame de Coeur and Ingrid Bergman vere evidenced as slightly attacked (SA); and the cultivars: Flamenco, Karla, Rumba, Maria Callas, Grand Mogol and Montezuma were evidenced as medium resistant (MR). Very sensible (V.S) leaves black spot attack proved to be the cultivars Mainzer Fastnacht, Rose Gaujard, Kordes Perfecta, Detroit, Horido şi Konigin der Rosen, but they can be used as indicators for this disease. At this group of cultivars the plants were premature defoliated at the end of July. The climatic conditions from the Black Sea coast, characterized by strong wind, high temperature during the day (26-28oC) and the presence of the water condense on the vegetative organs of the plants favorised, the occurrence of the powdery mildew attack produced by Sphaerotheca pannosa var rosae fungi starting with the last decade of May. The symptoms were noticed initially on the both sides of the leaves as irregular white dusty spots (Fig.2). The biological material for investigations was represented by 50 roses cultivars preserved in the collection owned by Research Station for Fruit Growing Constanţa. Observation were made regarding the attack frequency (F%) and intensity (I notes) of the pathogens Diplocarpon rosa, Sphaerotheca pannosa var rosae and Phragmidium mucronatum, and finally the attack degree (AD) was calculated For disease intensity (I notes) the scale „0-6” was used. The observations were carried out in the period of attack maximum for each of three key pathogens studied. According the attack degree (A.D.) value, the cultivars were classified in five resistance classes as follow: - resistant (R) cu A.D.= 0-5% - slightly attacked (SA) with A.D. = 5.0-12.5% - medium resistant (MR) with A.D. = 12.5-22.5% - sensible (S) with A.D. = 22.5-37.6% - very sensible (VS) with A.D. = more than 37,6% To establish the D.L., in order to establish the five classes of cultivars the average value took into calculations ws 22.9%. 3. Results and Discussions In 2008, the attack of Diplocarpon rosae, fungi which determine the black spot disease was evidenced on rose cultivars in the third decade of May, being stimulated by the amount of precipitation at the vegetation start (114,9 mm) and by the high atmospheric relative humidity (over 75%). The disease symptoms occurrence on leaves consisted in some black spots, between 2-5 mm up to 56 Marioara Trandafirescu et al. / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010) Table 1. Behaviour of some cultivars roses to the attack of the main pathogens agents CULTIVARS Brocade Grand premiere Creole Traviata Grand prix Horido Apogee Luchian First love Concerto Konigin der Rosen Foc de tabără Miss Univers Chicago Peace Kronenburg Bel Ange Cocotte Samurai Monica Montezuma Don Juan Grand Mogol Sutter’s Gold Effel Tour Detroit Maria Callas Simfonia albă Mabella Pascali Rumba King’s Ranson Baccara Superstar Kordes Perfecta Madame Meilland Coup Foudre Mr. Lincoln Dame de Coeur Rose Gaujard Carina Queen Elisabeth Eminance Mainzer Fastnacht Karla Flamenco Ingrid Bergman Ambassador Emerande d’or Parfum Rubin Diplocarpon rosae G.A. Resistan (%) -ce class 7.2 S.A. 0 R 12.4 S.A. 0 R 32.4 S 47.3 F.S. 0 R 0 R 1.2 R 3.7 R 63.0 F.S. Sphaerotheca pannosa var. rosae G.A. Resistan (%) -ce class 11.6 M.R. 0 R 6.5 S.A. 1.2 R 0 R 1.0 R 0.8 R 4.2 S.A. 17.6 M.R. 9.7 S.A. 14.2 M.R. Phragmidium mucronatum G.A. Resistan (%) -ce class 12.3 M.R. 4.6 S.A. 3.2 S.A. 14.6 M.R. 0 R 3.6 R 0 R 3.6 S.A. 38.4 F.S. 12.2 S.A. 0 R 4.1 26.3 32.0 34.6 4.3 0 1.6 0 17.4 3.6 15.0 11.7 0 37.9 15.6 24.0 57.0 12.2 19.3 0 6.3 1.2 51.2 0.7 R S S S S.A. R R R M.R. R M.R. S R F.S. M.R. S F.S. S.A. M.R. R S.A. R F.S. R 1.6 0 37.6 17.6 0 0 3.6 0 9.6 0 2.0 42.6 3.6 6.4 0 0 10.2 0 6.9 1.2 22.0 3.6 17.9 7.3 R R F.S. M.R. R R R R S.A. R R F.S. S.A. S.A. R R M.R. R S.A. R S R M.R. S.A. 1 0 22.8 0 0 18.3 4.3 0 4.5 0 42.6 53.2 1.2 0 1.2 1.2 13.7 0.9 22.0 54.6 7.2 12.6 21.3 4.6 R R S R R M.R. R R S.A. R F.S. F.S. R R R R M.R. R M.R. F.S. S.A. M.R. M.R. S.A. 0 2.6 7.2 42.6 12.4 3.2 1.2 38.6 R R S.A. F.S. S.A. R R F.S 12.6 0 12.6 51.8 6.3 1.2 2.6 7.9 M.R. R M.R. F.S. S.A. R R S.A. 17.2 0 42.1 22.4 1.2 0 0 9.6 M.R. R F.S. M.R. R R R S.A. 13.0 19.3 7.2 1.2 0.6 0 0 M.R. M.R. S.A. R R R R 1.6 3.6 14.6 6.2 38.6 1.8 0 R R M.R. S.A. F.S. R R 2.0 12.3 1.2 1.6 0 2.6 0 R S.A. R R R R R Fig. 2. Făinarea trandafirului – Sphaerotheca pannosa var. rosae Afterwards, the attack progressed covering almost entirely the leaves, which turn in yellow, then dried and fallen down. In this case the attack progressed also on the young floral buds of the sensible cultivars, which were covered by the mycelium felt and they could not open. Assessment of the data presented in the same table revel that, a high (natural) resistance to this damaging pathogen stroke manifested the cultivars: Grand Prix, Miss Univers, Maria Callas, Pascali, Simfonia albă, their vegetative organs were entirely clear from Sphaerotheca pannosa var rosae. fungi symptoms. At the other pole were the cultivars: Emerande d’or, Chicago Peace, Sutter’s Gold and Rose Gaujard which were rated as very sensible (V.S.), but they can be used as sensibility indicators. The attack produced by Phragmidium mucronatum fungi, was noticed at the end of the first decade of May and progressed until the last decade of September. In this month this pathogen attack frequency (F%) and the intensity (I notes) registered the highest values. From the beginning the disease progressed on all plants organs: leaves, young branches, stalks and floral buds (Fig. 3). On these organs was noticed the presence of some orange pustules representing the fungus ecidia. 57 Identification of some rose genitors with resistance... / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010) Fig. 3. Rugina trandafirului – Phragmidium mucronatum 5. In the last decade of May, on the inferior face of the plant leaves, small pale-yellow pustules occurs, representing the nest of uredospores which produce repeated secondary infections during the vegetation period. Starting with the second decade of June, on the inferior face of the plant leaves, was observed the presence of the black pustules representing the shelter of the teleutospores containing the resistance organs of the fungus. Among the cultivars that manifested a pronounced genetic resistance to the attack of this pathogen can be mentioned: Apogee, Bel Ange, Emerande d’or, Grand Prix, Kroenenburg, Kroningin der Roson, Miss Univers, Detroit, Rubin, Queen Elisabeth, Eminance. Their vegetative organs were entirely clear from pathogen signs all of the vegetation period. The vast majority of the other cultivar studied showed themselves as slightly attacked (SA) or medium resistant (MR). In the case of this pathogen, as was highlighted in table 1, very sensible cultivars (VS) manifested the cultivars Grand Mogol, Sutter’s Gold, King’s Ranson and First love. References [1] BEDIAN G., 1980. Rust (Phragmidium sp.) on roses, R.P.P., 59, 4, 1562. [2] BON Y., Bourdin J., Berthier G., 1978. Efficacité de quelques fongicides vis-á-vis de L’oidium du rosier (Sphaerotheca pannosa var. rosae), Phytiatrie – Phytopharmacie, 27 (3), 199-205. [3] CASTLENDINE P., Grout B.W.W., Roberts A.V., 1981. Cuticular resistance to Diplocarpon rosae, Transaction of the British Mycological Society, 47. [3] COSTACHE C., Costache M., Argatu Constanta, 1993. Rezultate preliminare privind comportarea unor soiuri de trandafir la atacul principalilor agenţi patogeni. Analele I.C.L.F. vol. XII, 119129. [4] HAGAN A. K., Gillian C. H., Fare D. C.,1987. Evaluation of new fungicides for control of rose black spot, Journal of Environmental Horticulture 6 (2), 67-69. [5] LÖSING H., 1988. Bekämpfung von Rosenrost, Deutsche Baumschule 40 (11), 518-519. [6] MORRISON L. S., 1978. Preliminary results on the evaluation of fungicides for the control of black spot of rose. Nursery Research Field Day P – 777, 59-60. [7] QVARNSTRÖM K., 1989. Control of black spot (Marssonina rosae) on roses, Växtskyddsnotiser 53 (3), 58-63. [8] PALMER L. T., Salac S. S., 1978. Reaction of several types of roses to black spot fungus, Diplocarpon rosae, Indian Phytopathology 30 (3), 366-368. [9] ROLIM P. R. R., Toledo A. C. D., Cardoso R. M. G., Brignani Neto F., Oliveira D. A., 1990. Comparison of fungicides for control of rose black spot (Diplocarpon rosae) and powdery mildew (Sphaerotheca pannosa var. rosae), Summa Phytopathologicals 16 (3-4), 269-274. [10] SAUNDERS P. J. W., 1970. The resistance of some cultivars and species of Rosa to Diplocarpon rosae Wolf causing black spot disease, Natn. Rose, A., 118-128. [11] SEMINA S. N., Klimenco Z. K., 1976. Evaluation of garden rose gene pool for 4. Conclusions In the Romanian zone of Black Sea coast, the pathogens with economical importance for the roses grown in open fields are: Diplocarpon rosae Wolf, Sphaerotheca pannosa (Wallr) Lev var rosae Woron şi Phragmidium mucronatum (Pers) Schlecht. The rose cultivars Emerald d’or, Bel Ange, Apogee, Foc de tabără, Queen Elisabeth, Rubin, Parfum and Rubin, present genetic resistance for all three damaging agents and can be used as resistance genitors in the works carried out to bread new disease resistant rose cultivars. The fact that under the some climatic conditions, the rose cultivars manifest various attack degrees to the pathogens reveals that, the resistance is cultivars trait, which represent the key factor in prevention of the most damaging specific pathogens. 58 Marioara Trandafirescu et al. / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010) resistance to powdery mildew. Byull Gosudar, Nikit. Bot. Sada, 2 (30), 48-54. [12] SIMONYAN S. A., 1973. Powdery mildew of rose in the Erevan Botanical Grden. Biol. J. Armenii, 26 (7), 62-73. [13] SZEKELY I., Wagner Şt., Drăgan Maria, 1981. Rezistenţa diferitelor soiuri de trandafir faţă de atacul de făinare (Sphaerotheca pannosa var rosae) în funcţie de unele caracteristici anatomomorfologice, Simpoz. CAER Cluj, ASAS, ICPP. [14] WAGNER Şt., Râureanu V., 1996. Principalele boli şi dăunători ai trandafirilor şi combaterea lor. Rosarium, nr. 1. 59 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 PRELIMINARY DATA ON MELEDIC – MANZALESTI NATURAL RESERVE (BUZAU COUNTY, ROMANIA) Daciana SAVA *, Mariana ARCUŞ**, Elena DOROFTEI * *Ovidius University, Natural Sciences Faculty, Aleea Universitatii No. 1, corp B, Constanţa, 900470, Romania, e-mail: daciana.sava@gmail.com ** Ovidius University, Faculty of Pharmacy, Aleea Universitatii No. 1, corp B, Constanţa, 900470, Romania __________________________________________________________________________________________ Abstract: the Meledic –Manzalesti Reserve is situated in the central-eastern part of Romania, in Buzau County, 60km north form town of Buzau. The Reserve (136 hectares) is delimitated by four rivers and it is situated at a medium altitude of 530 m. Because of the remarkable forms of relief, appeared as a result of dissolution of salt, the presence of a salt cave unique in Europe and of a number of lakes with fresh water, this area was declared in 1986 Geological and Speological Reserve. Later (in 2000) due to the presence of an interesting flora and fauna it was established the value of its natural heritage, and was declared as „Protected Natural Area” with geological, speological, floral and faunistic importance. In 2007 it was declared „Site of Community Importance” and will become area of special conservation after the validation of the European Commission. The present study took place over a period of two years, with field trips in various periods of the year. As for the flora, taxons belonging to over 100 genera were identified, Most genera belong to Fabaceae, Asteraceae, Labiatae, Rosaceae and Umbelliferae families. The statistical analysis showed as biological forms, the predominance of hemicrytophytes. As floristic elements, the Euro-Asiatic and Central European elements predominated. As regarding the ecological preferences (humidity, temperature and soil reaction) it has been observed the domination of xeromesophytic, mezothermal and euriionical species. Keywords: Natural Reserve, Manzalesti –Meledic Natural Reserve, Romania __________________________________________________________________________________________ 1. Introduction In 1978, a group of Romanian spelologists, part of “Emil Racoviţã” Speologists Club from Bucharest, discover in the sub-Carpathians Mountains at Mânzãleşti, the longest (300 m), the deepest (44 m) and the most ramificated cavity in salt in the country, second in the world as oscillation of level, third as length, wich they named “The cave with three entrances“ from Sãreni. In 1980 the “6s“ Cave from Mânzãleşti is discovered, the longest cave in salt world-wide at that moment (1257 m) and the second as oscillation of level (-32 m), with numerous ramifications. Later on, other galleries, were discovered of a total of 4257 m length, 32 caves digged in salt, taking the Mânzãleşti cave to the second place in the world for caves digged in carst of salt. Later on, in 1986, the salt carst from Mânzãleşti becomes a reserve of The Romanian ISSN-1453-1267 Academy from the geological and speleological point of view. According to the Habitat Directive 92/43/CEE concerning the conservation of natural habitats, wild flora and fauna, the protected areas network Natura 2000 appears in România, which includes also in this network the Meledic Plateau of Mãnzãleşti commune, Buzãu County, according to Law nr 5/5 March 2000 [1]. The site has the ROSCI 0199 code and is classified under category IV (according to UICN) as Special Conservation Area. The reserve is part of the Continental Biogeographical Region; its existance is trying to protect the “Ponto-sarmatic deciduous thickets” habitats. Characterization of the “Meledic Plateau” Reserve © 2010 Ovidius University Press Preliminary data on Meledic-Manzalesti Reserve... / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010) „Meledic Plateau” Reserve is situated in the subCarpathians Mountains, in the Lopãtari Dingle and in the Slãnic river superior basin (tributary streamof Buzãu river) (latitude N 45ο 29’ 49” and longitude of E 26ο 37’ 16”). The reserve has a length of 1.7 km, on the NorthSouth axis and 1.2 km on the West-East direction, holding a total surface of 157 hectares, the plateau being situated at an altitude between 400m and 600m. In the Eastern side, the delimitation is determined by the Jgheab river (a tributary stream of the Slãnic river), North of the Meledic stream and West of the Sãrat stream, the latter tributary stream of the Jgheab Valley, which emptyes into the Sãrat stream in turn. Out of these rivers and streams only the Meledic stream has fresh water, the other streams being also alimented with salty springs, their water having a brackish taste. Fig.2. Aspect of the abrupt slopes in Plateau Meledic Reserve (vest view) Relief The Meledic Plateau Slopes are very abrupt, allowing sometimes to see the structure of the plateau represented by a layer of clay and shale on the upper side, with a thickness of 10 to 30 meters, under which there is the block of salt, tall up to a few hundred meters. The Meledic Reserve represents one of the most unprecedented places, the relief is expanding on the salt located on the surface or shallow depth, resulting one of the most interesting regions in our country. A very diversified terrain in shape and size develops because of the dissolution of salt on slopes (Fig.1, Fig.2). On the western side we can notice blocks of salt integrated in clay and salty shale, on which gaps and limestones have developed, in comparison with the southern side where vein of salt can be noticed even on the surface. Where the salty water rivers come out on the surface arises a rapid vaporisation of the water resulting in especially beautiful salt cristals. The plateau is located on the upper side of the reserve and is crossed by sinkholes, closed dingles, oval or round, with a diameter that can reach sometimes 40 m and a depth of 25 m, wider dingles results by their blending. On the bottom of such sinkholes, where the salt was covered with a denser layer of clay, freshwater lakes were formed, receiving water only from rain or snow meltdown. These lakes have karstic origins, all the underground springs are salt watered, the connection with these ending long time ago. The presence of freshwater lakes on a salt massif is considered a unique phenomenon. Soils In the Meledic reserve we encounter a large variety of soils. On the steep slopes, where the salt layers are very close to the surface or even on the surface, we find the white alkali. On the slopes where the water carries small amounts of silt the vertisols are formed, on heavy clay rocks (with high clay content). On the plateau we meet halomorphous soils, which have a high content of soluble salt, that occur on Fig.1. Aspect of the abrupt slopes in Plateau Meledic Reserve (south view) 62 Daciana Sava et al. / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010) partially covered with sediment surfaces. A great diversity of halophile species develop here. In sinkholes where the sedimented clay have allowed the formation of lakes, the soils are hydromorphic, formed due to an excess of moisture, which can be permanent or temporary. In areas where the water has a permanent stagnation, the pseudogleic soil is formed. The plant species developing here are thermophilic. In forested areas we can find reddish brown forest soils, especially clay soils. In these areas we especially find Pontic or Mediterranean species. Climate The sub-Carpathians Mountains have a temperate-continental climate, with regional differences imposed by the shape of relief, but also by the position at the intersection of climate influences northwest, eastern and southern. Being located in a lowland area of the subCarpathians, the Meledic Plateau has a low hill climate with a tendency of aridity in summer. The average annual temperatures fit between 6ο C and 8ο C. The average annual temperature of the coldest month, January, is of 3ο C, and of the hottest month, July, is of 18ο C. The average annual rainfall is around 700-800 mm. The largest amount of rainfall is in May and June, and the driest months are September and October. 2. Material and Methods Field trips have been organized for the flora studies in the Meledic Reserve from Mânzãleşti: two trips in the months of May and June (months that have a rapid vegetation development), and in the months of April, July, August, September only one trip, through the years 2007-2008 to catch the different stages of vegetation (vernal and estival). After rating, we made up a floristic list, in which the plants have been placed in the right systematic units [1, 2, 3]. Based on this list, we made out: the systematic analysis of the vegetation, the bioform spectrum, the geoelements spectrum, the spectrum for ecological preferences: humidity, temperature and soil reaction [4, 5]. 3. Results and Discussions Due to the field trip a total of 133 taxa were registered. The following taxa were identified in the study area: Acer campestre L. Ph (MM); Eur.; U 2,5 T 3 R 3, Achillea millefolium L. H; Euras.; U 4 T 3 R 0 , Adonis aestivalis L. Th; Euras; U 3 T 4 R 3, Agrimonia eupatoria L. H; Euras.; U 2,5 T 3 R 4, Ajuga genevensis L. H; Euras.; U 2,5 T 3 R 4 , Alisma plantago-aquatica L. HH; Cosm.; U 6 T 0 R 0, Alnus incana (L) Moench Ph (MM); Eur.;U 4 T 2 R 4 , Alnus viridis (Chaix.) DC Ph (MM);Alp.-eur;U 3,5 T 2,5 R 3, Anchusa officinalis L. limba boului); TH; Eur.; U 2 T 3,5 R 0, Anemone nemorosa L. G; Eur; U 3,5 T 4 R 0, Anemone ranunculoides L. G; Eur; U 3,5 T 3 R 4 , Artemisia vulgaris L. H; Circ.;U 3 T 3 R 4, Astragalus onobrychis L. H; Euras.; U 1,5 T 3,5 R 4,5, Ballota nigra L. Th; Centr. Eur.); U 3 T 3,5 R 0, Betonica officinalis L .(Stachys officinbalis L.) H; Euras.; U 3 T 3 R 3, Brassica rapa L.Th; Med; U 3 T 3 R 4, Campanula rapunculoides L. H; Euras.; U 3 T 2 R 0, Capsela bursapastoris Medicus Th; Cosm; U 3 T 0 R 0, Carex digitata L. H; Euras.; U 3 T 3 R 3, Carum carvi L. TH; Euras.; U 3,5 T 3 R 3, Centaurea spinulosa Roch. H; Centr. Eur.; U 2,5 T 0 R 3, Centaurea nervosa Willd. H; Alp.-eur.;U 3 T 0 R 3 ; Centaurium umbellatum Gilib. Th; Centr.eur.;U 3 T 3 R 2, Chaerophyllum bulbosum L. TH; Centr. Eur; U 4 T 3,5 R 4,5, Chrysanthemum leucanthemum L. H; Euras.; U 3 T 3,5 R 3, Chrysanthemum corymbosum L. H; Euras.; U 3 T 3 R 3, Clematis vitalba L. Ph ; Centr. Eur.; U 3 T 3 R 3, Colchicum autumnale L. G; Eur; U 3,5 T 3 R 4, Coronilla varia L. H; Centr. Eur.; U 2 T 3 R 4, Cornus mas L. Ph (M); Pont. medit.; U 2 T 3,5 R 4, Cornus sanguinea L. Ph (M);Centr. Eur); U 3 T 3 R 4, Corylus avellana L. Ph (M); Eur.; U 3 T 3 R 3, Crataegus monogyna Jacq. Ph (M); Euras.;U 2,5 T 3 R 3, Cytisus hirsutus L. Ph (N); Centr. Eur.; U 2,5 T 3 R 2, Daucus carota L. TH; Euras.; U 2,5 T 3 R 0, Delphinium consolida S.F.Gray (Consolida regalis) Th; Euras; U 3 T 4 R 4, Diplotaxis muralis L. Th; Centr. Eur; U 2,5 T 3,5 R 4, Draba verna Chevall Th; Euras; U 2,5 T 3,5 R 0, Echium vulgare L. TH; Euras.; U 2 T 3 R 4 , Elaeagnus angustifolia L. Ph (M); Euras; U 0 T 3 R 4,5, Epipactis atropurpurea Raf G; Euras.; U 2 T 0 R 4,5, Equisetum arvense L. G.; Cosm.; U 3 T 3 R 0, Erigeron canadensis L. Th; Adv.; U 2,5 T 0 R 0, Eryngium campestre L. H; Pont. medit.; U 1 T 5 R 4 , Euphorbia cyparissias L. H; Eur.; U 2 T 3 R 4 , Euphrasia rostkoviana Hayne. Th; Centr. Eur.; U 3 T 3 R 3, Fagus sylvatica L. Ph (MM); Centr. Eur.; U 3 T 3 R 0, Festuca pratensis Hudson H; Euras; U 3,5 T 0 R 0, Ficaria verna L. (Ranunculus ficaria Huds.) H;Euras: U 3,5 63 Preliminary data on Meledic-Manzalesti Reserve... / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010) T 3 R 3 , Filipendula ulmaria Maxim. H; Euras: U 4,5 T 2 R 0, Fragaria viridis Duch. H; Euras.; U 2 T 4 R 3, Fraxinus ornus L. Ph (M); Medit.; U 1,5 T 3,5 R 5, Gagea pratensis Dumort. G; Eur; U 2 T 3 R 3, Galanthus nivalis L. G; Centr. Eur.; U 3,5 T 3 R 4, Galium verum L. H; Euras.; U 2,5 T 2,5 R 0, Galium vernum Scop. H; Euras; U 3 T 2 R 2, Hippophae rhamnoides L. Ph (M); Euras.; U 0 T 3 R 4,5 , Hypericum perforatum L. H; Euras.; U 3 T 3 R 0, Juniperus communis L. Ph (M); Circ.; U 2 T 0 R 0 , Knautia arvensis (L.) Coult. H; Eur.; U 2,5 T 3 R 0, Knautia silvatica Duby. H; Centr. Eur; U 2 T 3 R 0, Larix decidua Miller Ph (MM); Carp; U 2,5 T 0 R 0, Lathyrus tuberosus L. H; Euras.; U 2 T 4 R 4 , Lathyrus pratensis L. H; Euras; U 3,5 T 3 R 4, Leontodon hispidus L. H; Euras.;U 2,5 T 0 R 0 , Lepidium draba Desv. H; Euras: U 2 T 4 R 4, Linum austriacum L. H; Euras.; U 1,5 T 3,5 R 4 , Lithospermum purpureo-caeruleum L. H; CentrEur;U 2 T 3,5 R 4, Lythrum salicaria L. H; Circ.; U 4 T 3 R 0 , Medicago lupulina L. Th ; Euras.; U 2,5 T 3 R 4, Medicago falcata L.Th; Euras.; U 2 T 3 R 4, Melampyrum arvense L. Th; Eur.; U 2 T 3,5 R 4,5 , Melilotus officinalis (L.) Pallas Th; Euras.; U 2,5 T 3,5 R 0, Morus nigra L. Ph (MM); Adv; U 2 T 3,5 R 4, Muscari comosum (L. ) Miller G; Eur.; U 1,5 T 3,5 R 0, Myosotis sylvatica Hoffm. H; Euras: U 3,5 T 3 R 3, Onobrychis viciifolia Scop.H; Euras.; U 2 T 3 R 0 , Orchis purpurea Huds. G; Centr. Eur.;U 2,5 T 4 R 4,5, Origanum vulgare L. H; Euras.;U 2,5 T 3 R 3 , Orlaya grandiflora L. Th; Med; U 2 T 3,5 R 4, Ornithogalum refractum Kit. G; Balc-Pan- Cauc; U 2 T 3,5 R 4, Phragmites australis Steudel HH; Cosm.; U 5 T 0 R 4, Picea excelsa Link Ph. (MM); Centr. Eur.; U 0 T 0 R 0, Pinus sylvestris L. Ph.(MM); Euras.; U 0 T 0 R 0 , Plantago media L. H; Euras; U 2,5 T 0 R 4,5, Poa pratensis L. H; Circ: U 3 T 0 R 0, Polygala amara L. H; Eur.; U 0 T 2 R 4,5 , Polygala major Jacq. H; Pont. –medit.; U 2 T 3 R 4,5, Potamogeton natans L. HH; Cosm.; U 6 T 2,5 R 4 , Potentilla reptans L. H; Cosm; U 3,5 T 0 R 4, Potentilla argentea L. H; Euras; U 2 T 4 R 2, Primulla officinalis Hill. H; Euras.;U 3 T 2 R 5, Prunella vulgaris L. H ; Circ. U 3 T 3 R 0 , Prunella grandiflora (L.) Scholler H; Eur.; U 3 T 3 R 4,5, Prunus cerasifera Ehrh. Ph (M); Euras: U 2 T 4 R 0, Pyrus piraster Burgsd. Ph (M); Eur; U 2 T 3 R 4, Quercus dalechampii Ten. Ph (MM); Medit; U 2,5 T 3 R 0, Ranunculus arvensis L. Th; Euras.; U 3 T 3 R 0, Rhinanthus minor L. Th; Eur.; U 3 T 0 Ro , Rosa canina L. Ph (N); Eur.; U 2 T 3 R 3 , Rubus caesius L. Ph (N); Eur.; U 2 T 3 R 4 , Salix alba L. Ph (MM); Euras; U 5 T 3 R 4 , Salix caprea L. Ph (M); Euras; U 3 T 3 R 3, Salix pentandra L. Ph (MM); Euras.;U 4,5 T 0 R 3,5, Salvia verticillata L. H; Medit.;U 2 T 4,5 R 4, Salvia nemorosa L. H; Centr. Eur.; U 2,5 T 4 R 3 , Scabiosa ochroleuca L. H; Euras.; U 2 T 4 R 4, Schoenoplectus tabernaemontani (Gmelin) Palla HH;Euras.; U 5,5 T 4 R 5, Senecio vernalis Waldst et. Kit. Th; Euras.;U 2,5 T 4 R 0, Silene vulgaris Garke H; Euras: U 3 T 3 R 4, Sinapis arvensis L. Th; Euras.; U 3 T 4 R 4, Sisymbrium sophia Webb. Th; Euras; U 2,5 T 4 R 4, Stachys lanata Jacq. H; Medit.; U 2 T 0 R 0, Thlaspi perfoliatum L. Th; Euras; U 2,5 T 3,5 R 4,5, Thymus glabrescens Willd. Ch; Pont.-pan.; U 2 T 4 R 0 , Tilia cordata Miller Ph (MM); Eur.; U 3 T 3 R 3, Tragopogon pratensis L. H; Euras.; U 3 T 2 R 3, Trifolium campestre Schreb. Th; Eur; U 3 T 3 R 0, Trifolium medium L. H; Euras.; U 3 T 3 R 0, Typha angustifolia L. HH; Circ.; U 6 T 4 R 0, Ulmus laevis Pall. (velniş); Ph (MM); Eur.; U 4 T 3 R 3, Veronica chamaedrys L. Ch; Euras.; U 3 T 0 R 0 , Veronica arvensis L. Th; Eur; U 2,5 T 3 R , Veronica teucrium L Ch; Euras.; U 1,5 T 4 R 4,5, Vicia angustifolia L. Th; Euras.; U 2 T 3 R 0, Vicia sepium L. H; Euras.; U 3 T 3 R 3, Vicia cracca L. H; Euras; U 3 T 0 R 3, Vicia hirsuta S.F. Gray. Th; Euras; U 2.5 T 3,5 R 4, Viola arvensis Murr. Th; Cosm.; U 3 T 3 R 0, Viola hirta L. H; Euras.; U 2 T 3 R 4, Viola tricolor L. Th; Euras.; U 2,5 T 3 R 0. Statistic flora analysis Taxa found in the reserve belong to 4 classes, 43 families and 133 species. The largest number of species has the following families: Fabaceae (16 species), Labiatae, Rosaceae and Compositae (10 species each), Scrophulariaceae, Umbelliferae (5 species each), the other families were represented by only 1, 2, or 3 species each. Analysis of the biological forms Analyzing the spectrum of the biological forms we discover that in the reserve hemicryptophyte dominate (41%) from the species identified. These are followed (21%) by the phanerophytes which together with therophites (24%) form another 43% from the biological forms, the rest being represented by the geophytes and the helohydrophytes (Fig.3). 64 Daciana Sava et al. / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010) are 42% from the total of the identified species in the reserve, these being found in the droughtiest places, specially on the meadows. Notable are also the mesophilic (U 3 -U 3,5 ) which have a percentage of 37%, and can be found in areas where the light is scarce or there is an excess in humidity, where the swamps dry up during the summer but nevertheless have an excess in moisture. The xerophilic (U 1 -U 1,5 ) can be found in 5% and this shows the hot and arid summer climate, being especially noticed on the slopes that have a south exposure, covered with a small seam of clay soil. In a percentage of 6%, the mesophilical (U 4 -U 4,5 ) species that prefer soils from humid to moist-wet, are found near lakes or where there is an excess in moisture all year long. Remarkable is the presence of the hydrophilic (U5U 5,5 ) and ultrahydrophilic (U 6 ) species which together form 5% from the total of species, and can be found in the ponds or on their border where water is present all year round. The amphytoletant (U 0 ) species can also be found in the reserve in a percentage of 6%, being the most adaptable for these special conditions (Fig.5). 4% 20% 41% 4% 8% 2% 21% H Ph Ch G HH Th TH Fig.3. Analysis of the biological forms Analysis of the floristic elements The analysis of the floristic elements mark out the dominant euro Asiatic elements, which among those central European sum up approximately 82 species (70%) from the reserve flora, forming more than half the floral elements which means that they constitute the floristic background of this reserve. Mediterranean and Ponto Mediterranean floristic elements, which are thermophile species found especially on the sunny slopes, form together 8%. However, the cosmopolitic species are also remarkable, representing 5% from the total species found in the reserve. The fact that the reserve in situated in sloppy area is confirmed by the presence of the circumpolar and even alpine European at the reserve level, together representing 7% of the total species (Fig.4). 1% 1%1% 5% 5% 20% 22% 4% 1% 14% U3 Daco.Balc Cosm Alp 9% 2% 46% 5% Carp. Adv Pont Pan 1%2% 28% U3,5 U4 U2,5 U5 4% 2%1% 2%2% 5% U5,5 U6 U0 U1 U1,5 U2,5 U2.5 14% Balc.Pan. Cauc Eur Medit Fig. 5. Ecological spectrum of humidity Circ Centr.Eur Euras Temperature Mesothermal (T3-T3,5) appear in a percentage of 63%. Mild thermophilic (T4-T4,5) species appear in a percentage of 12%, which suggests that the climate in the reserve in a temperate-continental one.The amphylotolerant (T0) appear in 16% of the total. The cryophilic (T1) species are missing, and the microthermal (T5) species appear in a small percentage, only 1% (Fig.6). Fig.4. Analysis of the floristic elements Ecologic study of the cormophytes Humidity If we group the plants by their humidity regimen in which they are adjust to live here, we will discover that the most dominant are xeromesophilic (U 2 -U 2,5 ) which 65 Preliminary data on Meledic-Manzalesti Reserve... / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010) 45% 45% 2% 6% 14% 17% T3 plants mostly found in the northern regions, with an arid climate, and are presented in the reserve through the annual or biannual species. Phanerophytes (21%) presented by trees and scrubs, indicate the presence of forests in the reserve, as well as the cover of the slopes with scrubs which assure its stabilization. The analysis of the floristic elements reveals the predominant Euro-Asian elements, which along the central European totalize approximately 82 species (70%) from the reserve flora, representing the floristic background of the reserve. Floristic Mediterranean and Ponto Mediterranean elements, which are theomophilic species, can be found on the sunny slopes. The fact that the reserve in situated in a hill area can also be acknowledged because of the T3,5 14% 1% 1% T4 T4,5 T5 T0 T2 T2,5 T3 Fig.6. Ecological spectrum of temperature Soil reaction 37% of the identified species are euroionic (R0); poor acid-neutrophilic species (R4-R4,5) are presented in the same proportion; acid-neutriphilic species (R3-R3,5) can be found in a percentage of 21%, and the neutrophilic-basophilic (R5) are found only in a percentage of 3%. The balance of the acidophilic plants (R2) is just 2%, and those highly acidophilic (R1) are missing (Fig.7). 3% circumpolar and even alpine European species found here. The presence of xeromesophitic species (U 2 -U 2,5 ) in a percentage that represents almost half of the total of the identified species in the reserve (42%), indicate a arid climate which is specially caracteristical for the medows. The mesophilic species (U 3 -U 3,5 ), which have a pretty high percentage (37%), can be found in the areas where the light is scarce or it is excessively moisturized or in the areas where the swamps completely dry out during summer, but remain excessively moisturized. The mesothermal (T 3 -T 3,5 ) along the mild thermophilic (T 4 T 4,5 ) are presented in a higher percentage, which mean that the climate in the reserve is a temperate continental one. Regarding the distribution according to the reaction of the soil, the euroionic species (R 0 ) can be found in a pretty high percentage (34%), almost equal to those of the species and the poor acid-neutrophilic (R 4 - R 4,5 ) (40%). Remarkable is also the presence of the acid-neutrophilic species (R 3 - R 3,5 ) (21%) which have a percentage that is worth taking into account; the presence in the reserve of different types of habitats: rivers (with fresh and salt water), lakes, meadows, forests. Studies are to be done in the future to analyze the interesting and diverse flora of the region. 20% 34% 1% 2% R3 30% 10% R3,5 R4 R4,5 R5 R0 R2 Fig. 7. Ecological spectrum for soil reaction 4. Conclusions The flora in the reserve is highly diversified, being represented by the distribution of the species in 43 families, predominant being the families Fabaceae, Labiatae, Compositae. Hemicryptophyte (41%) appear in the highest percentage indicating the presence of the herbal evergreen species, adaptable to the edapho-climatic conditions in the areas. Therophites (20% + 4%) are 5. References [1] MOHAN GHE, ARDELEAN A., GEORGESCU M., 1993 - Rezervaţii şi monumente ale naturii din România, Casa de Editurã şi Comerţ, București, 201 pp. 66 Daciana Sava et al. / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010) [2] BELDIE AL., 1977- Flora României Determinator ilustrat al plantelor vasculare, vol. III, Editura Academiei R.S.R, 406 pp. [3] CIOCÂRLAN V., 2000 - Flora ilustratã a României, Editura Ceres, Bucureşti, 1138 pp. [4] DONIŢÃ N., IVAN D., 1975 - Metode practice pentru studiul ecologic şi geografic al vegetaţiei: 112-331, Editura Didacticã şi Pedagogicã, Bucureşti. [5] SANDA V., POPESCU A., DOLTU I., DONIŢÃ N., 1983 - Caracterizarea ecologicã şi fitocenologicã a speciilor spontane din flora României, “Ecological and phytocoenologyical characterisation of the spontaneous species in Romanian flora” in: Nat.Scienc.Suppl. 25, Stud.Communic. Muz. Brukental, Sibiu, 126 pp. 67 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 CONTRIBUTIONS TO THE BIOMETRICAL AND PHYTOBIOLOGICAL STUDY ON WILD GARLIC Mariana LUPOAE*, Dragomir COPREAN*, Rodica DINICĂ**,Paul LUPOAE*** * Ovidius University Constanţa, Faculty of Natural and Agricultural Sciences Street Mamaia, nr. 124, Constanţa, 900527, România, mariana_lupoaie@yahoo.com ** Dunărea de Jos University Galaţi, Faculty of Science, Street Domnească no. 47, 800008, Galaţi, rodinica@ugal.ro *** Natural Sciences Museum Complex Galaţi-Botanical Garden, Street Regiment 11 Siret no. 6A, 800340 Galaţi, paul_lupoae@yahoo.com _____________________________________________________________________________________ Abstract: The purpose of our study was the biometrical and phytobiological analysis of leafs and bulbs on wild garlic. This species grows spontaneously in the Romanian flora and was harvested for obtaining the drugs on Măcin Mountains (Luncavita Forest), at altitudes of 150÷200m. By macroscopic examinations in different phenophasis established in the area study extended population with Allium ursinum L. ssp. ucrainicum Kleopow et Oxner (Fam. Alliaceae). The biometrical calculation have been performed according to the literature, early spring, in months february, march, april and may of year 2010. Leaves finesse expressed by l/L is different: march l/L=32÷37%; april l/L=22÷30%; may l/L=16÷28%. Language leaf mature surface is between 71,24÷145,2 cm². Average mass bulbs = 2,4 g/buc and length by 12 mm to 50 mm. Keywords: wild garlic; Allium ursinum L. subsp. ucrainicum; leafes and bulbs; biometry. __________________________________________________________________________________________ 1. Introduction The Allium genus includes approximately 500 species spread worldwide. Allium ursinum L. is a monocots on family Alliaceae and is widely in Europa, Asia Minor, Caucasus, Siberia up to the Kamchatka Peninsula. In Romania this species ”mezohigrofita” grows in frequent clusters at the shadows of the trees. It has elliptical-lanceolat leaves with white flowers grouped and from the biochemical point consist through the presence of the ether oils with sulfur, that are giving their own smell [1-3]. Under various popular names- buckrams, wild garlic, broad-leaved garlic, wood garlic, sremuš or bear's garlic- this species is used by locals in preparations for spring salad and is very appreciated for many qualities. They have been shown to have applications as antimicrobial, antithrombotic, antitumor, hypolipidaemic, antiarthritic and hypoglycemic agents [4-8]. ISSN-1453-1267 The last researches about the population of A. ursinum from Romania put in evidence differences of biomass depending of the geographic area and the local pedoclimatic conditions. Wild garlic is a plant which grows on soils with high mineral trophicity and takes place into the “megatroph” category with the value V= 85-100 % [9]. The opportunity of this biometrical and phytobiological studies consist in the representation of some morphologic-bulbus,folium,flores-by wild garlic elements harvested from the Luncavita Forest (Macin Mountains), a plant with high pharmaceutic potential. 2. Material and Methods The harvesting of the bilological material that was realized with the agreement of the O.S. Macin that manages the area of the Luncavita Forest (U.P.I.”Izvorul lui Gavrila”). © 2010 Ovidius University Press Contribution to the biometrical and phytibiological.../Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010) The macroscopic exam served as a review of the observed characters with free eye or with the magnifer and as sensory through the perception of the smell and the taste on the informations contained in the bibliography of speciality and own researches [10, 11]. The biometrical observations were obtained based on the published biometrical calculation methods. The surface of the leaves was measured with the help of a mathematic model by the summing of the geometrical figures distributed uniformly on an sample of 30 leaves [12]. Some representative examples indentified on the area are stored in the Herbarium of Botanical Garden Galati and of the Pharmacy and Medicine Faculty of “Dunarea de Jos” University Galati. Fig. 1. The escape of the wild garlic through the wood fragments ( original photo) Biometric analysis consists of the following items (Table 1): the leaf length (L), the leaf width (l), the petiole length (Lp), the percentage ratio-leaf finesse (l/L), number ribs (R), mass of green leafs (M). On the bulbs (Table 2, Fig. 4) were measured the length (L), the mass (M), diameter (D) and number of the roots (No.roots). Our biometric studies made on the leaves of A.ursinum shou that the rapport between the width and the length of the limb leaf is conversely proportional with the procedure of growing (Fig.3):in March l/L=32÷37%; in April l/L=22÷30%; in May l/L=16÷28%. The form of the leaves at the immature plant from March is predominant ovat-eliptical and at mature in May is elliptical lanceolat. The growing in length of the petiole is more pronounced in April 104÷280mm. The arch parallel nervatiune is numerical constant in all of the phases. The surface of the limb leaf mature is contained between 71,24÷145,2 cm² and the weight of the green leaves is 24,49 g/10 mature leaves. So it can be confirmed that the foliar biomass of the population of A.ursinum from Luncavita Forest is lower by comparison with the morphological studies on the same harvestes species from Botosani area ( 35,85 g/10 leafes) [8]. 3. Results and Discussions A.ursinum grows on big areas in the Luncavita Forest only in north hills or near the water. Sometimes this species can be found in other zones but in low population. The acompaining flora is composed by different species like: Corydalis solida, Asarum europaeum, Corylus avellana, Tilia tomentosa, Hedera helix, Polygonatum latifolium, Scilla bifolia, Carpinus betulus, Viola odorata, Ranunculus ficaria, Lamium purpureum, Galium aparine, Geum urbanum, Anthriscus cerefolium, Muscari botryoides and others. Our observations show us that under the shrubs (Corylus avellana) known for its organic requestsrich soils,deep,loose- and a rich litter,wild garlic reaches the base of the shrubs [14]. Also,the power of growing and penetration of the wild garlic was noticed even through the woody,halfdescomposed fragments (Fig. 1). Simple bulbs or two united can be found at a relative depth small in the soil (3-5cm) especially in the humus layer and they have good developed roots and branched by 3÷15cm length (Fig.2). The appearance of the leaves are leveled:first in March, the second simultaneous with the third (by case). The most of the plants have two leaves. 68 Mariana Lupoae et al. / Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010) Table 1. Biometrical elements on wild garlic leafes Months/ Leaf number 1 2 m 3 a 4 r 5 c 6 h 7 8 9 10 1 2 3 a 4 p 5 r 6 i 7 l 8 9 10 1 2 3 m 4 a 5 y 6 7 8 9 10 L mm l mm Lp mm l/L % R no M g 145 120 150 110 125 100 148 143 135 149 200 210 157 168 205 195 155 188 190 209 261 185 250 260 240 187 180 259 227 235 50 45 50 35 44 35 49 50 45 50 60 60 35 39 59 59 35 57 57 60 70 40 70 71 67 41 39 70 37 38 98 81 100 74 81 75 100 100 88 100 256 280 104 106 280 280 105 270 268 280 359 193 360 360 343 192 195 358 324 325 34 37 33 32 35 35 33 35 33 33 30 28 22 23 28 30 22 30 30 29 26 22 28 27 27 21 22 27 16 16 18 17 19 17 17 17 18 19 17 19 22 22 17 17 22 22 17 22 22 22 23 17 23 23 19 17 17 23 18 18 0,71 0,56 0,8 0,57 0,6 0,5 0,75 0,7 0,69 0,79 1,48 1,5 1,04 1,09 1,4 1,35 1,01 1,23 1,26 1,42 2,8 2,31 2,62 2,9 2,38 2,32 1,9 2,79 2,18 2,29 Fig. 2. Bulbus with radix on wild garlic (original photo) Table 2. Biometrical elements on wild garlic bulbs Months/ Bulb number 1 f 2 e 3 b 4 r 5 u 6 a 7 r 8 y 9 10 The harvesting of the bulbs was realized in February before the entry in vegetation of the plants. In the studied area the identified bulbs had different sizes (Fig.4): max.length=50mm with the diameter D=7mm; min. length =12mm with the diameter D=3mm. The number of roots is contend between 7÷10. The medium mass of the bulb is 2,4 g/piece. L mm M g D mm No. roots 30 14 25 50 40 13 20 35 12 15 3,3 1,2 3,1 4,5 3,8 1,1 1,3 3,6 1,1 1,2 5 4 5 7 7 3 5 6 3 4 8 7 7 10 10 8 7 9 7 7 Fig. 3. The percentage ratio-leaf finesse wild garlic Legend: S1-sample march; S2-sample april;S3sample may 69 Contribution to the biometrical and phytibiological.../Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010) The informations from literature of speciality about the determination of underspecies of Allium ursinum are very little because of the similarities between underspecies ursinum and underspecies ucrainium. Even, the difference can be realized when the plants reach the level of inflorescence. With the help of the magnifier can be observed that the pedicels don’t prezent papillae and they have a smooth surface (Fig.6) characteristic of the underspecies ucrainicum [1]. Fig. 4. Measurements of bulbs on wild garlic The infloresecense is umbeliform arranged on a florifera strain that passes the height of the leaves. The floral stalk leaves from the same place take a vaulted form. At the base of the stalks there are bacterias wich form an involucres. The flower is type 3, specific mococotyledonous, and the tricarperal ovary crushed emits a specific smell of the garlic and has a sweety taste wich attracts the bugs (Fig.5). Fig.6. “Pediceli” and ovary “tricarpelar” of A. ursinum (original photo) The spreading of the ursinum underspecies, includes areas from Mountains Macin -Greci, Tiganca, Niculitel- but there is not specified the area of the Luncavita Forest [2]. Also, the recent studies realized in North Dobrogea show on the Gymnospermio-Celtetum Association the presence of the Allium ursinum species but there aren’t any references about the underspecies [13]. 4. Conclusions Fig.5. Inflorescence of A. ursinum Our studies realized in Luncavita Forest (O.S. Macin, U.P. I, “Izvorul lui Gavrila”) shows the presence of the wild garlic on large areas but only on north hills near water. The literature informations of speciality are confirmed concerning the exigency of the species against trophicity of the soil and our observations shows an affinity of the wild garlic by Corylus avellana. From organoleptic point of view there has been seen the next things: all of the vegetal products harvested-roots,bulbs,leaves,flowers-they have a piquant taste and powerful smell of garlic; the roots have second branches and the bulbs (white-yellow) are sourrounded by white and transparent membranes; the green leaves on the both faces are elliptical lanceolat and the pedicels are smooth, that means the determination ucrainicum Kleopow et Oxner [1,2]. 70 Mariana Lupoae et al. / Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010) We found only an foliar dimorphism in the first fenophase in March when the report l/L is high 32÷37% opposite the values from May l/L=16÷28%. The mass of the leaves is 24,49 g/10 the values of the leaves is lower comparative with the population of the wild garlic from other zones (Botosani) and the mature bulbs grow until 50mm length with a mass about 2,4g. The harvested vegetal products-bulbs,flowershave a specific smell of garlic. The fitobiological and biological analysis permitted us an identify in premiere, of the subspecies studied from the Macin Mountains (Luncavita Forest) and that would be: Allium ursinum L. ssp. ucrainicum Kleopow et Oxner. The investigation of the natural population is necesarry because of the biosintetical potential therefore it can be influented by de pedoclimatic conditions from the area of the sampling of the plants. The studies undertaken by us can offer the premise of the harvest,conservation and processing of some vegetal products from wild garlic in order to improve the farmocognostic researches. [6] STAJNER D., POPOVIC B.M., CanadanovicBrunet J., Stajner M., 2008. Antioxidant and scavenger activities of Allium ursinum, Fitoterapia 79, p. 303-305. [7] ARHANA SENGUPTA et al., 2004. Allium Vegetables in Cancer Prevention: An Overview, Asian Pacific Journal of Cancer Prevention, Vol 5: 237-245. [8] MIHĂILESCU R., Mitroi G. Iacob E. Miron A., Stănescu U., Gille E. , Creţu R., Ionescu E. Giurescu C. , 2008. Obtaining of phytoproducts for the cardiovascular diseases profilaxy, Note 1 Some investigation of the Allium ursinum chemical composition , The 5’th Conference on Medicinal and Aromatic Plants of Southeast European Countres , BRNO. [9] CONSTANTIN D. CHIRITA et al., 1964. Fundamentele naturalistice si metodologice ale tipologiei si cartarii stationale forestiere , Editura Academiei RPR, pag.110-113 . [10] *** FARMACOPEA ROMANA, 1993. Ediţia a-X-a , Editura Medicală Bucureşti , pg. 10-63. [11] BUCUR L.,ISTUDOR V. et al., 2002. Analiza farmacognostica, Instrument de determinare a identitatii puritatii si calitatii produselor vegetale, Editura Ovidius University Press, Constanta, pg. 7-87. [12] BERCU R., BAVARU A., 2007. Biometrical and morpho-anatomical observations on Acer monspessulanum L. (Aceraceae) leaves, Contributii Botanice, XLII, Gradina Botanica “Alexandru Borza” Cluj Napoca, pg. 105-110. [13] PETRESCU M. Cercetări privind biodiversitatea unor ecosisteme forestiere din Dobrogea de Nord, Editura Nereamia, Napocae-Tulcea, pg.61-72, 2004. [14] NEGULESCU E., SAVULESCU Al., 1957. Dendrologie, Editura Agro-Silvica de Stat, Bucuresti, pg. 184-188. 5. References [1] CIOCÂRLAN V., 2000. Flora ilustrată a României–Pteridophyta et Spermatophyta, Editura Ceres, Bucureşti, pg. 919-925. [2] SĂVULESCU T., Flora Republicii Socialiste România, Editura Academiei Republicii Socialiste România , 1966, Vol. XI, pg.193-266. [3] TITA I., 2005. Botanica farmaceutica editia a IIa, Editura Didactica si Pedagogica Bucuresti, pg. 854-863. [4 ] DJURDJEVIC L.,, Dinic A., Pavlovic P., Mitrovic M., Karadzic B., Tesevic V., 2003. Allelopathic potential of Allium ursinum L., Biochemical Systematics and Ecology 32, pg.533544. [5] ONCEANU (LUPOAE) Mariana, Miron Tudor Lucian, Dinica Rodica. Studiul unor principii active din specia Alium ursinum recoltată din flora spontană, publicat în rezumat, Conferinţa Naţională a Societăţii Ecologice din România, Galaţi, octombrie 2009. 71 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 DINITROPHENYL DERIVATIVES ACTION ON WHEAT GERMINATION Cristina Amalia DUMITRAS -HUTANU*, *„Al. I. Cuza” University of Iasi, 11 Carol I, Iasi-700506, Romania, hutanu_amalia@yahoo.com __________________________________________________________________________________________ Abstract: Several dinitrophenyl ethers such as 2,4-dinitroanisol, 2,4-dinitrophenetol, 2,4-dinitro-1(octadecyloxy) benzene, 3-(2,4-dinitrophenoxy)propane-1,2-diol or other similar compounds have been synthesized and tested comparatively to some well-known metabolic inhibitors and stimulators within the germination experiments. As a result, the weight of the resulted plantlets was diminished by 2,4-dinitroanisol and 3-(2,4-dinitrophenoxy)propane-1,2-diol treatments (1.15 g/lot and 32.03 mg/plantlet in the case of 2,4dinitroanisol; 0.11 g/lot and 22.3 mg/plantlet in the case of 3-(2,4-dinitrophenoxy)propane-1,2-diol). Dinitrophenyl ethers inhibited seed germination, most probably by blocking oxidative phosphorylation. A novel mechanism of action of these pesticides was discussed. Consequently,the toxicity processes of these pesticidelike compounds and metabolic inhibitors was discussed in direct relationship with their infrared absorbance and fluorescence quenching. Keywords: pesticide toxicity, dinitrophenyl ethers, dintirophenols, wheat germination. __________________________________________________________________________________________ 1. Introduction Dinitroderivatives, especially the aromatics, are frequently used as intermediates in the manufacture of pharmaceuticals, dyes, pesticides and explosives. They have multiple biological actions, being used as insecticides, fungicides, herbicides and acaricides [1, 2]. However, Environment Protection Agency in SUA (EPA) included the dinitrophenols on the list of national priorities and in concentration of 3-46 mg dinitrophenol/kg body kill; no antidote is known (max. admissible dose 70 ppb in water, EPA, 2004). It is assumed that dinitrophenols hinder the proton translocation through the mitochondrial inner membrane and therefore oxidative phosphorylation is inhibited (ATP is no longer formed and the cells deprive of essential energy supply). It is also possible that the dinitrophenols act toxically due to the inhibition of formation of some triplet states (instable biradicals) by a resonance process with the triplet structures in the living cells (A. Szent-Gyorgyi-Nobel Prize, 1957) [3, 4, 5, 6, 7, 8]. Because the existing data are inconclusive and do not support a precise action mechanism of dintrophenyl derivatives on living organisms, it was necessary to synthesize some ISSN-1453-1267 dinitrophenols and dinitrophenyl ethers whose biological activity should be tested. The purpose of this paper is to compare the biological activity of some synthetic compounds containing the di- and nitrophenyl moiety with that of some well-known metabolic inhibitors and stimulators. Because germination experiments are easy, cheap, fast and spectacular, the testing of the action of some action of some known and newly synthesized substances on living organisms will be performed using germinating cereal seeds [3-5]. The possible mechanism of toxicity of these chemicals and pesticides are discussed in the light of the biostructural theory by Eugen Macovschi as well as the chemiosmotic theory by Peter Mitchell [6, 7, 8]. 2. Material and Methods Biological material. The wheat samples (Triticum aestivum), Henika variety, were taken from the Agricultural Research Station in Suceava. The 1000 seeds weighed 37.2 g and had a residual humidity of 12%. Chemical reagents. The reagents used were of analytic purity (Merck, Sigma, Chimopar) and the solution and the water slurries were prepared using redistilled water. Thus, © 2010 Ovidius University Press Dinitrophenyl derivates action on wheat germination / Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010) dinitroderivatives such as 2,4-dinitrophenetol, 2,4dinitroanisol, 3-(2,4-dinitrophenoxy)propane-1,2-diol and 2,4-dinitrophenyl-glutathione were synthesized. Several solutions of dinitrophenyl ethers and dinitrophenols with the concentrations 4x10-3 M were prepared. A blank with bidistilled water was also carried out. Equipment. The chemical syntheses were carried out using the organic chemistry lab equipment of the Chemistry Department of “Al. I. Cuza” University of Iasi. The experiments and the germination determinations were performed in Petri dishes, on double Watmann no. 1 filter paper at room temperature. The separation and purification of the compounds obtained were carried out using thin layer chromatography on silica gel (Kieselgel 60F 254 , Merck) and on silica gel column. The infrared spectra were taken on a Jasco FT/IR660Plus Fourier spectrometer in the range from 0 to 15000 cm-1. Procedure. The germination parameters were measured according to ISTA recommendations (Seed Science and Technology, 1993), however we worked also with lots of 50 seeds which were laid to germinate on filter paper, in Petri dishes, in three repetitions. The first count took place after three days (energy of germination, EG), the second after 7 days (germination rate, GR). The germinated, abnormal and dead seeds as well as the resulting plantlets were counted. The treatment lasted for an hour, followed by the distribution of the seeds uniformly in the Petri dishes, on double filter paper, together with the treatment solution. The seeds with a visible root were considered germinated. The seeds were watered daily with 5 ml of redistilled water. The plantlets were cut at the level of the seeds 7 days after, measured and weighed (height, H, in cm and mass, m, in grams). Statistics. The results were processed using the Tukey test [9]. The mean square deviation s x of the samples was also calculated, as well as t factor, with a view to compare the results obtained under the action of different treatments. phenylalanine by 6.3% the average mass of plantlets as compared to the blank. 2,4-Dinitrophenol inhibited total the germination process of wheat seeds, (Table 1). 1 2 3 4 5 6 Fig. 1. The biological effect of some nitrophenyl derivatives and other compounds on wheat germination. 1 – Blank (water); 2 – DNP; 3 – DNG; 4 – DNA; 5 – resorcinol; 6 – L-β-phenylalanine. Table 1. The toxicity of 2,4-dinitrophenol (DNP), 3(2,4-dinitrophenoxy)propane-1,2-diol (DNG), 2,4dinitroanisol (DNA), resorcinol and L-βphenylalanine and at concentrations 4x10-3 M in a wheat seeds germination experiment. GermiPlantlets Average nation of roots size *) Treatment Rate mass (S, cm) (G. R.) (m, mg) 3. Results and Discussion 1 - Blank (water) 2 - DNP, 4x10-3 M 3 –DNG, 4x10-3 M 4 – DNA, 4x10-3 M 5– resorcinol, 4x10-3 M 6 – L-βphenylalanine, 4x10-3 M 90% 6.4+0.6 18.7+1.3 0% 0 0 42% 2.5+0.6 8.6+3.2 85% 4.6+0.6 13.9+0.1 83% 5.6+0.7 18.9+0.2 87% 6.0+0.8 19.1+0.6 D (Tukey test) 4.3 1.3 1.2 The height of the plantles treated with 2,4dinitrophenol (DNP), 3-(2,4-dinitrophenoxy)propane-1,2-diol (DNG), 2,4-dinitroanisol (DNA), resorcinol and L-β-phenylalanine and at As for the stimulative effect, the most active substance in these experiments proved to be resorcinol, which increased slightly by 5.5% and 74 Cristina Amalia Dumitras - Hutanu /Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010) concentrations 4x10-3 M, as compared to blank (water) treatment. It is apparent from the table that the root mass treatments with resorcinol and L-βphenylalanine is higher than the control samples. Lot A Lot B Lot C Ave rage 12 10 Number of plantlets 8 Number of plantlets Blank Lot A Lot B Lot C Ave rage DNA 10 6 4 2 8 0 40 6 60 80 Plantle ts siz e (mm) 4 2 Fig. 4 – The height of the plantlets of the lots treated with 2,4-dinitroanisol. 0 40 60 80 100 120 Plantle ts siz e (mm) Resorcinol Fig. 2. The height of the plantlets of the lots blanck Lot A Lot B Lot C Ave rage 10 Lot A Lot B Lot C Ave rage DNG 3 Number of platlets Number of plantlets 8 6 4 2 2 1 0 40 0 20 30 40 50 60 60 80 100 Plantle ts siz e (mm) 70 Plantlets size (mm) Fig. 3 . The height of the plantlets of the lots treated with 3-(2,4-dinitrophenoxy)propane-1,2-diol) Fig. 5. The height of the plantlets of the lots treated with resorcinol. 75 Dinitrophenyl derivates action on wheat germination / Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010) L-β-phe nylalanine At concentrations of 3x10-3 M, DNP does not allow the germination of wheat seeds The toxicity mechanism of these pesticides, pesticide-like compounds and metabolic inhibitors may be discussed in direct relationship to their infrared absorbance and fluorescence quenching (not shown). Thus, all of them have a significant absorbance at about 6000 cm-1 in IR, corresponding to ∆G of ATP formation (as previously shown by G. Drochioiu, personal communication) and quench the fluorescence of tryptophan and other biological compounds. Fluorescence quenching of tryptophan (1µg/µl) was tested using 2,4-dinitro-ortho-cresol (DNOC). The intense quenching activity of DNOC was associated with a stronger uncoupling property. According to Drochioiu’s hypothesis, one must take into consideration the fact that the pH modification could be a secondary phenomenon, being possible to transfer the energy of triplet states to the ADP molecule, which incorporates it as ATP. The transition from an excited state to a normal state leads to the release or absorption of a proton, depending on the acid or base character of the compound that is in an excited state. 2,4-Dinitrophenols act as uncouplers of the breathing from oxidative phosphorylation, which results in an intensified oxygen consumption, without ATP synthesis. Dinitrophenols normally disturb ATP production within the cellular mitochondria, because the ATP is the molecule which stores and supplies energy for cellular activities [6-8]. The present theories, such as P. Mitchell’s chemiosmotic theory, claim that, unlike the other enzymes in the mitochondrial respiratory chain, the ATP pumps protons from the intermembrane space towards the matrix. Thus, the energy that the other enzymes in the chain use to accumulate protons in the intermembrane space is recuperated. This energy is necessary for the ADP phosphorylation reaction with the mineral phosphate, in the presence of Mg ions, the reaction being endothermic and requires more than 31 kJ/mol. Present research showed that it is possible for the dinitrophenyl ethers to act in a non-chemical way, probably through radical and triplet status formation, and that the proton translocation could be a secondary phenomenon in the process of oxidative phosphorylation. The action mechanism of the compounds investigated is concordant with Eugen Lot A Lot B Lot C Ave rage 16 14 Number of plantlets 12 10 8 6 4 2 0 40 60 80 Plantle ts siz e (mm) 100 Fig. 6. The height of the plantlets of the lots treated with L-β-phenylalanine Of all the five figures can be seen as only for seedlings lengths blank if the three lots are very close. In other cases the range of lengths of seedlings was higher. These differences show once again disrupting the development of seedlings in the presence of chemicals, whether stimulatory or inhibitory effect. Toxicity 2.4-Dinitrophenol 100 80 60 40 20 0 0 2 4 6 8 10 Concentration (mM) Fig. 7 – The biological effect of 2,4-dinitrophenol on wheat germination. Toxicity seen in this figure is that of 2,4dinitrophenol (DNP). 76 Cristina Amalia Dumitras - Hutanu /Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010) ninitrophenyl ethers. Roum. Biotechnol. Lett. , Vol. 14(6), 4893-4899 pp. [6] LEHNINGER A. L., 1987 - Biochimie, Ed. Tehnică, Bucureşti, Vol. 2, 473, 547 pp. [7] DROCHIOIU G., 2006 - In Life and mind. In search of the physical basis. S. Savva (ed.) Trafford Publ., Canada, USA, Ireland & UK, 43 pp. [8] MITCHELL P., 1978 - David Keilin’s respiratory chain concept and its chemiosmotic consequences, Nobel Lecture. [9] SNEDECOR G. W., 1994 - Statistical methods applied to experiments in agriculture and biology, The Iowa Stat Univ. Press, 255 pp. Macovschi’s well-known biostructural theory, but contradicts Peter Mitchell’s chemiosmotic hypothesis. 4. Conclusions Development of seedlings is disrupted in the presence of chemicals, whether stimulatory or inhibitory effect. Dinitrophenyl ethers and phenolic derivatives displayed a similar pattern of biological activity inhibiting seed germination, most probably by blocking oxidative phosphorylation. Therefore, we discussed a mechanism of biological activity as well as that of toxicity related to the energy transfer in biological systems to form ATP. The proton translocation through the biological membranes could be a secondary phenomenon, but the most important event in the toxicity process of dinitrophenyl derivatives. Further research is still necessary to clarify the specificity of the biological activity of diand nitrophenols. 5. References [1] BEWLEY J.D., Black M., 1994 - Seeds, Physiology of development and germination, Plenum press, 2nd Ed., New York and London. [2] COMĂRIŢĂ E., Şoldea C., Dumitrescu E., 1986 - Chimia şi tehnologia pesticidelor, Ed. Tehnică, Bucureşti, 188 pp. [3] DUMITRAS-HUTANU, C. A., Pui, A., Drochioiu, G., 2008 - Dinitrofenil derivati cu posibile aplicatii in medicina si biologie: mecanisme de actiune si toxicitate, Materiale si procese innovative. Simp. V, ZFICPM, Iasi, Editura Politehnium, 61-66. [4] DUMITRAŞ-HUŢANU C. A., Pui, A., Gradinaru, R., and Drochioiu, G., 2008 Toxicity of dinitrophenyl derivatives used as pesticides and their environmental impact, Lucrări ştiinţifice USAMV Iaşi, seria Agricultură, 51. [5] DUMITRAŞ-HUŢANU C. A., Pui A., Jurcoane S., Rusu E., Drochioiu G. 2009 - Biological effect and the toxicity mechanisms of some 77 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 THE ACTION OF SOME INSECTICIDES UPON PHYSIOLOGICAL INDICES IN RANA (PELOPHYLAX) RIDIBUNDA Alina PĂUNESCU*, Cristina Maria PONEPAL*, Octavian DRĂGHICI*, Alexandru Gabriel MARINESCU* * University of Pitesti, Faculty of Science, Departament of Ecology Târgu din Vale Street, no.1, Pitesti,410087, Romania, alina_paunescu@yahoo.com __________________________________________________________________________________________ Abstract: The goal of this work is to study the physiological changes induced by the action of three insecticide (Carbetox, Actara 25WG and Reldan 40EC) in Rana (Pelophylax) ridibunda. The animals used in the experiment were divided in four experimental lots: two lots of control individuals (first lot was kept at 4-6ºC and the second lot at 22-24ºC) and two experimental lots in which the animals were treated with toxic substance and kept at 46ºC, respectively at 22-24ºC. The toxic was administrated with intraperitoneal shots (one shot every two days, in a scheme of three weeks). At the end of the experiment we determinate number of erythrocytes (RBC), leukocytes (WBC) and glycemia values. We observe a decrease in number of blood cell (RBC and WBC) as well as an increase a glycemia values. Keywords: Carbetox, Actara 25WG, Reldan 40EC, frog, erythrocytes, leukocytes, glycemia __________________________________________________________________________________________ 1. Introduction A number of factors have been suggested for recently observed amphibian decreases, and one potential factor is pesticide exposure. The use of pesticides in agriculture can have effects on amphibian within or adjacent to application areas [1, 2]. Aside from direct deposition or drift, insecticides can reach aquatic habitats via runoff, which depends on precipitation, soil conditions, and slope of the catchments area [3]. The effect of insecticides on large aquatic organisms varies with the test organism. Frogs were found to be more sensitive and may serve as a biological indicator for pesticide contamination in waterways [4]. Our goal is to study the effect of three insecticides (Carbetox, Actara 25WG and Reldan 40EC) in some physiological parameters (number of erythrocytes and leukocytes, glycemia level) in Rana (Pelophylax) ridibunda at two heat level (4-6ºC and 22-24ºC). Carbetox (malathion) is one of the most widely used organophosphorous pesticides with numerous agricultural and therapeutic applications, and exposure to environmentally applied malathion can lead to adverse systemic effects in anurans. Cutaneous absorption is considered a potentially ISSN-1453-1267 important route of environmental exposure to organophosphorous compounds for amphibians, especially in aquatic environments [5]. It is slightly toxic via the dermal route. Actara 25WG is a neonicotinoid insecticide active against a broad range of commercially important sucking and chewing pests and it has as its component the major active ingredient, thiamethoxam (25%). Thiamethoxam's chemical structure is slightly different than the other neonicotinoid insecticides, making it the most water soluble of this family. Reldan 40EC is an insecticide from the class of organophosphates. The active substance of this insecticide is chlorpyrifos. 2. Material and Methods Adult specimens of amphibians (Rana ridibunda), of both sexes, captured in spring (AprilMay) from the surrounding areas of the city Pitesti (Romania) were kept unfed in freshwater aquaria. The water was changed daily to avoid the accumulation of toxic substances. After 10 days of adaptation in the lab, when they were unfed, the frogs were separated in lots, which were used separately for the following experiments: two lots of control individuals, containing animals kept in laboratory at 4-6oC, respectively at 22-24ºC with no treatment, in © 2010 Ovidius University Pres The action of some insecticides.../ Ovidius University Annals, Biology-Ecology Series 14: 79-82 (2010) running water which was changed everyday, (1) one lot containing animals which were subjected to treatment with insecticide and kept at 4-6ºC, (2) a second lot containing animals which were subjected to treatment with insecticide and kept at 22-24ºC. The toxic was administered by intraperitoneal shots, one shot every two days, in a scheme of 3 weeks. The administered dosage of insecticide was not lethal as none of the subjects died through the experiment. We used three different types of insecticide: Carbetox (active substance is malathion) in a dose of 0.01ml/g body weight, Actara 25WG (active substance is thiamethoxame) in a dose of 0.4mg/g body weight and Reldan 40EC (active substance is chloropyrifos-methyl) in a dose of 0.01ml/g body weight. The number of erythrocytes and leukocytes was microscopically determined with a Thoma cells numbering chamber, by using a small amount of blood collected from the heart [6]; the glycemia level has been determinate using an Accutrend GCT. hemoglobin, the number of erythrocytes, leukocytes and platelets in fishes were exposed to LC50 of some insecticides for seven days. As shown in Figure 1, as compared to the values recorded for the control individuals of frog, the number of erythrocytes increases by 51.14% for the animals which were treated with Reldan 40EC in a dose of 0.01 ml/g of body weight and kept at 4-6ºC, while animals treated with the same concentration of Reldan 40EC but kept at 22-24ºC the number of erythrocytes increases by 76.88%. Increased number of erythrocytes under the action of Reldan 40EC has also been noticed by Păunescu et al [9]. 1000000 851000 691888.9 900000 number of erythrocytes/ml blood 800000 700000 600000 481111.1 4-6º C 500000 400000 22-24º C 358166.7 298400 225608.2 300000 301077.8 232405.6 200000 3. Results and Discussions 100000 0 The number of erythrocytes in the frog individuals subjected for three weeks to treatment with 0.01ml/g body weight of Carbetox was significantly affected as shown in Figure 1. The difference between the number of erythrocytes which was determined for the control and the ‘treated’ lot at 4-6ºC, an average decrease of 16.68% was found in the treated frog individuals who seemed to be related to the intense hemolytic activity. At 22-24ºC we registered a decrease with 37.01% in a number of erythrocytes. In animals treated with Actara 25WG in a dose of 0.4mg/g body weight, there has been a decrease in the number of erythrocytes with 35.11% to the control value for specimens kept at 4-6°C and with 37.42% for animals treated with insecticide and kept at 22-24°C. We mention that similar results in the number of erythrocytes in the lake frog were obtained by other researchers in similar experimental conditions in fish. Thus, Ponepal [7] found a decrease in the number of erythrocytes in fish under the action of Actara 25WG insecticide, as well as a decrease in the oxygen consumption. Also, Dhembare [8] recorded decreased control Carbetox Actara 25WG Reldan 40EC Fig. 1. The influence of some insecticide upon number of erythrocytes in Rana (Pelophylax) ridibunda The number of leukocytes (Fig.2) at the two heat levels registered similar changes to that of the number of red blood cells as can be seen in Figure 1. Carbetox insecticide in a dose of 0.01ml/g body weight determined a decrease in number of leukocytes with 87.26% as compared with the witness value, at 4-6ºC. An intensive leucopenia was also registered at 22-24ºC, when the number of leukocytes decreases with 101.93%. The number of leukocytes decreases by 28.52% to the witness for animals treated with Actara 25WG and kept at 4-6°C, while the value of this index is lower, 62.06% as compared to the witness, at higher temperatures (22-24ºC). Reldan 40EC in a dose of 0.01 ml/g of body weight was also affected the number of leukocytes. As shown in figure 2, the difference between the number of leukocytes which was determined for the control kept at 4-6ºC and the ‘treated’ lot kept at the same temperature, an average 80 Alina Păunescu et al. / Ovidius University Annals, Biology-Ecology Series 14: 79-82 (2010) decrease of 53.07% was found in the treated frog individuals. Similar results were obtained at 22-24ºC when the numbers of leukocytes decrease by 68.81% of the control value. Similar effects have been carried out by [10] studying the effects of chloropyrifos on mice. of animals kept at 4-6ºC and with 173.59% in the case of animals kept at 22-24ºC. These changes occur due to inhibition of glucose tissue by the toxic, and inhibition of Krebs cycle and glicolise enzymes, this leading to accumulation of glucose in the blood. 90 700 516.3333 62.16667 80 600 500 60 mg glucosis/ml blood number of leukocytes/ml blood 57.5 70 423.9444 400 4-6ºC 303 22-24ºC 300 226.3889 209.9444 195.8889 198.9444 4-6º C 22-24º C 34.83333 40 25.38889 30 161 200 50 20.66667 22.72222 18 20 11.16667 100 10 0 0 control Carbetox Actara 25WG Reldan 40EC control Fig. 2. The influence of some insecticide upon number of leukocytes in Rana (Pelophylax) ridibunda Carbetox Actara 25WG Reldan 40EC Fig. 3. The influence of some insecticide upon glycemia in Rana (Pelophylax) ridibunda The glycemia level was found to be significantly influenced by Carbetox insecticide. Thus, as shown in Figure 3, at a concentration of 0.01ml/g body weight, this index increases after three weeks of treatment to 61.29% of the control value at 4-6ºC. The same concentration of this toxic determinate, at 22-24ºC an increase of blood glucose concentration with 212.09%. It has been reported in several studies that hyperglycemia is one of the side effects in poisoning by OP in subchronic exposure and in acute treatment [11, 12, and 13]. Several studies have demonstrated some evidence for damage in pancreatic exocrine function after anticholinesterase insecticide intoxication [14, 15, 16, and 17]. The stimulation of pancreatic secretion secondary to cholinergic stimulation seems to be responsible for the development of pancreatitis [18, 19, 20, and 21]. The influence of Actara 25WG is also felt in the glucose level, whose values are shown in Figure 3. Its analysis shows an increase of glucose by 85.07% compared to witness for animals kept at a temperature of 4-6°C and treated with a concentration of 0.4mg/g Actara 25WG and 153.05% for the animals kept at 22-24°C and treated with the same concentration of toxic. Reldan 40EC in a concentration of 0.01ml/g body weight determinate, after three weeks of treatment, an increase of glycemia level with 127.41% as compared to the witness value in the case 4. Conclusions Analyzing comparatively the influence of three insecticides (Carbetox, Actara 25WG and Reldan 40EC) upon some physiological indices in Rana (Pelophylax) ridibunda, we found that these decrease (in percentage) the number of erythrocytes and leukocytes and increase the glycemia values. Only Reldan 40EC insecticide causes an increase in RBC. On the other hand, the toxic effect of these insecticides was proven to be more powerful at 2224ºC than 4-6ºC. 5. References [1] BERRILL M, BERTRAM S, PAULI B, 1997 Effects of pesticides on amphibian embryos and larvae. In: Green DM (ed) Amphibians in decline: Canadian studies of a global problem. Reports from the declining amphibian population task force. Herpetol Conserv, 1:233–245. [2] GREULICH K, HOQUE E, PFLUGMACHER S, 2002 - Uptake, metabolism, and effects on detoxication enzymes of isoproturon in spawn and tadpoles of amphibians. Environ Toxicol Saf, 52: 256–266. 81 The action of some insecticides.../ Ovidius University Annals, Biology-Ecology Series 14: 79-82 (2010) [3] LIESS M, SCHULZ R, LIESS MHD, ROTHER B, KREUZIG R, 1999 - Determination of insecticide contamination in agricultural headwater streams. Wat Res, 33: 239–247. [4] CALUMPANG SMF, MEDINA MJB, TEJADA AW, MEDINAJR, 1997 - Toxicity of Chlorpyrifos, Fenubucarb, Monocrotophos, and Methyl Parathion to fish and frogs after a Simulated Overflow of Paddy Water. Bull. Environ. Contam. Toxicol., 58: 909-914. [5] WILLENS S, STOSKOPF M, BAYNES R, LEWBART G, TAYLOR S, KENNEDYSTOSKOPF S, 2006 - Percutaneous malathion absorption by anuran skin in flow-through diffusion cells. Envtl. Toxicol. & Pharm, 22: 263-267. [6] PICOŞ CA, NĂSTĂSESCU GH, 1988 - Lucrări practice de fiziologie animală. Tipografia Universităţii din Bucureşti, Bucureşti, 107, 122123, 192-195. [7] PONEPAL MC, PĂUNESCU A, DRĂGHICI O, MARINESCU AlG, 2006 - Research on the changes of some physiological parameters in several fish species under the action of the thiametoxame insecticide. In: Proceedings 36th International Conference of IAD: 163-167. [8] DHEMBARE AJ, PONDHA GM, 2000 Hematological changes in fish, Punctius sophore exposed to some insecticides. Journal Experimental Zoo India, 3(1): 41-44. [9] PĂUNESCU A, PONEPAL CM, DRĂGHICI O, MARINESCU AlG, 2009 - The influence of Reldan 40EC insecticide upon physiological indices in Rana ridibunda. Lucrări Ştiinţifice USAMVB Seria B, LIII: 173-178. [10] AMBALI S, AKANBI D, IGBOKWE N, SHITTU M, KAWU M, AYO J, 2007 Evaluation of subchronic chlorpyrifos poisoning on hematological and serum biochemical changes in mice and protective effect of vitamin C. The Journal of Toxicological Sciences, 32: 111-120. [11] GUPTA PK, 1974 - Malathion induced biochemical changes in rats. Acta Pharmacol. Toxicol., 35(3): 191–194. [12] RODRIGUES MR, PUGA FR, CHENKER E, MAZANTI MT, 1986 - Short term effect of malathion on rats’ blood glucose and on glucose utilization by mammalian cells in vitro. Ectotoxicol. Environ. Safety, 12 (2): 110–113. [13] MATIN MA, HUSAIN K, 1987 - Cerebral glycogenolysis and glycolysis in malathiontreated hyperglycaemic animals. Biochem. Pharmacol., 36(11): 1815–1817. [14] GOKEL Y, GULALP B, ACIKALIN A, 2002 Parotitis due to organophosphate intoxication. J. Toxicol. Clin. Toxicol. J., 40(5): 563–565. [15] PANIERI E, KRIGE JE, BORNMAN PC, LINTON DM, 1997 - Severe necrotizing pancreatitis caused by organophosphate poisoning. J.Clin. Gastrenterol., 25: 463–465. [16] DRESSEL TD, GOODALE RL, ARNESON MA, BORNER JW, 1979 - Pancreatitis as a complication of anticholinesterase insecticide intoxication. Ann. Surg., 189: 199–204. [17] LANKISCH PG, MULLER CH, NIEDERSTADT H, BRAND A, 1990 - Painless acute pancreatitis subsequent to anticholinesterase insecticide (parathion) intoxication. Am. J. Gastroenterol., 85: 872–875. [18] KANDALAFT K, LIU S, MANIVEL C, BORNER JW, DRESSEL TD, SUTHERLAND DE, GOODALE RL, 1991 - Organophosphate increases the sensitivity of human exocrine pancreas to acetulcholine. Pancreas, 6: 398–403. [19] GOODALE RL, MANIVEL JC, BORNER JW, LIU S, JUDGE J, LI C, TANAKA T, 1993 Organophosphate sensitizes the human pancreas to acinar cell injury: an ultrastructural study. Pancreas, 8: 171–175. [20] WEIZMAN Z, SOFER S, 1992 - Acute pancreatitis in children with anticholinesterase insecticide intoxication. Pediatrics, 90: 204–206. [21] MORITZ F, DROY JM, DUTHEIL G, MELKI J, BONMARCHAND G, LEROY J, 1994 - Acute pancreatitis after carbamate insecticide intoxication. Intens. Care Med., 20: 49–50. 82 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 CHANGES OF SOME PHYSIOLOGICAL PARAMETERS IN PRUSSIAN CARP UNDER THE ACTION OF SOME FUNGICIDE Maria Cristina PONEPAL, * Alina PĂUNESCU*, Alexandru Gabriel MARINESCU*, Octavian DRĂGHICI* * Universitatea din Piteşti, Facultatea de Ştiinţe Str. Tg. din Vale, nr.1 Piteşti, România, e-mail: ponepal_maria@yahoo.com __________________________________________________________________________________________ Abstract: This study was carried out to analyze the effects of sublethal and lethal concentrations of Bravo 500 SC, Champion 50 WP, Tilt 250 and Tiradin 70 PUS fungicide on some physiological parameters (oxygen consumption, breathing frequency, number of erythrocytes) of the prussian carp (Carassius auratus gibelio Bloch). The acute and subacute toxicity of fungicides was evaluated in glass aquaria under semi-static conditions. Keywords: prussian carp, fungicide, Bravo, Champion, Tilt, Tiradin, breathings frequency, oxygen consumption __________________________________________________________________________________________ 1. Introduction The commercial product Bravo 500 SC is a concentrated suspension of chlorothalonil (500g / l); chlorothalonil (2,4,5,6 tetrachlor isophthal-nitrile) is a contact fungicide with curative and preventive action (works by stopping germination and the development of spores) for combating a large number of pathogens (leaf spots, downy mildews, alternarioses, fruit rots, brown tor of fruit, scab) that threaten the main crops [1]. The fungicide is part of group IV of toxicity; it is not toxic to bees, warmblooded animals and moderately toxic to insects [2]. Chlorothalonil and its metabolites are very toxic to fish, aquatic invertebrates and marine organisms [3]: LC50 (96 h) is of 0.25 mg/l for rainbow trout (Salmo gairdneri), 0.3 mg/l for sun perch (Lepomis macrochirus), 0.43 mg/l for sea devil (Ictalurus punctatus), etc. Champion WP (copper hidroxide) is a fixed copper fungicide widely used for control of fungal and bacterial pathogens. Copper is highly toxic in aquatic environments and has effects in fish, invertebrates, and amphibians, with all three groups equally sensitive to chronic toxicity [4]. The Champion WP product is toxic to fish and aquatic organisms (96-hour LC 50 Bluegill: 180 mg/l, 96-hour LC 50 Rainbow trout: 0.023 mg/l and 48-hour EC 50 Daphnia: 0.065 mg/l). Tilt 250 (the active ingredient is propiconazole – triazole fungicide) has protective, curative and ISSN-1453-1267 systemic activity. Propiconazole's mode of action is demethylation of C-14 during ergosterol biosynthesis, and leading to accumulation of C-14 methyl sterols. The biosynthesis of these ergosterols is critical to the formation of cell walls of fungi [5]. The propiconazole is non toxic for bees, invertebrates and soil bacteriae, but is dangerous for fish and ather aquatic organisms (LC50 values ppm for freshwater fish species: bluegill 1.3-10.2, brown trout 3.5, rainbow trout 0.9-13.2, carp 6.8-21.0, catfish 2.0-5.1 and fathead minnow 7.6) [6] , [7]. Tiradin fungicide (the active substance is the thiuram - tetramethylthiuram disulphide TMTD) is a general use contact fungicide with protective action, third group of toxicity. Dithiocarbamates form a large group of chemicals that have numerous uses in agriculture and medicine [8]. It is used to control Botrytis on fruit and vegetables and in seed treatment. The 96-hour EC50 for algae growth inhibition is approximately 1 mg/l (1 ppm), the 48hour EC50 for Daphnia is less than 0.21 ppm and the 96-hour LC50 for fish is approximately 0.1 ppm (Bluegill sunfish, 0.0445 mg/l, Rainbow trout, 0.128 mg/l and 4 mg/l carp) [9]. This study was carried out to analyze the effects of sublethal and lethal concentrations – of some fungicide: Bravo 500 SC (from 0.078125 x 10-3 to 12.5 x 10-3 ml/l water), Champion 50 WP (from 0.003 to 3 mg/l water), Tilt 250 (from 0.25 to 4 ml/l water) and Tiradin 70 PUS (from 0.01 to 0.16 ml/l © 2010 Ovidius University Press Changes of some physiological parameters… / Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010) water) on some physiological parameters (oxygen consumption, breathing frequency, number of erythrocytes 0.078125 x 10-3 and 1.5625 x 10-3 ml Bravo/l water, 0.003 mg Champion/l water and 1 ml Tiradin/l water) of the prussian carp (Carassius auratus gibelio Bloch). The acute and subacute toxicity of this fungicide was evaluated in glass aquaria under semistatic conditions. II.1 – fish subjected to Bravo 500 SC in concentrations of 0.00078125, 0.0015625, ml /l water and the control lot II.2 – fish subjected to Champion 50 WP in concentrations of 0.003 mg/l water and the control lot II.3 - fish subjected to Tilt 250 in concentration 1ml /l water and the control lot The fungicides concentrations were determined by preliminary tests of survival. The introduction of fish in solutions was done after their mixing and aeration for 5 minutes. The water temperature was 16-18°C, the "immersion" solution was changed every 24 hours, and aeration of water was continuous; the fish were not fed during experiments to avoid further intervention of this factor [10]. The testing method was systematic with refreshing solution at 24 hours after the calculations of the day, in aquariums of 100 l (50 l, respectively) for each experimental lot. Determination of oxygen consumption was done by means of the oximetre and Winkler method and erythrocytes were counted with Thoma chamber, using a small amount of blood from the caudal artery on the optic microscope [10], [11]. The statistical interpretation of the results was performed with ANOVA (LSD) test. 2. Material and Methods Determinations were made between January 2004 and October 2009 on prussian carp samples (Carassius auratus gibelio Bloch), captured from the surrounding rivers of Piteşti. Animals were acclimatized for 10 days before the completion of experiments in aquariums with a capacity of 100 l and 50 l [10], under conditions of natural photoperiodism, a period in which they were fed once a day (ad libitum), at around 10 am. After acclimatization in the laboratory, the fish were separated in two experimental variants (lots of 10-20 fish - average weight 18 g) subjected to fungicides. I. Determinations of oxygen consumption and frequency of respiratory movements at intervals of 24, 48, 72, 96, 168 and 336 hours on all samples of these lots (depending on survival) on prussian carp subjected to: - I.1. Bravo 500 SC in concentrations of 0.00078125, 0.0015625, 0.003125, 0.0625, 0.0125 ml /l water and the control lot - I.2. Champion 50 WP in concentrations of 0.003, 0.03, 0.3 and 3 mg/l water and the control lot Tilt 250 in concentrations of 0.25, 0.5, 1, 2, 4 ml /l water and the control lot - I.3. Tiradin 70 PUS in concentrations of 0.01, 0.02, 0.04, 0.08, 0.16 ml /l water and the control lot II. Hematological determinations (after one, respectively two weeks of exposure to the fungicide, the fishs were sacrificed to achieve intakes of blood necessary to hematological calculations (number of erythrocytes). 3. Results and Discussions The first four figures (fig.1-4) shows the average frequency of the respiratory movements of prussian carps exposed to the action of some fungicide (Bravo, Champion, Tilt and Tiradin). 84 Maria Cristina Ponepal et al./ Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010) Fig.1. The influence of Bravo fungicide upon breathing frequency on prussian carp Fig.4. The influence of Tiradin fungicide upon breathing frequency on prussian carp Bravo and Champion have changed the respiratory rhythm of prussian carps in all investigated concentrations. For all concentrations tested the effect of the fungicide is initially stimulating and inhibitory as regards the frequency of respiratory movements. In two experimental variants (0.01 and 0.02 ml/l water)Tiradin is stimulating of the breathing frequency of fish; at the concentration of 0.04, 0.08 and 0.16 ml/l water, the fungicide caused a decrease in the respiratory rhythm of prussian carps. Changes of prussian carps oxygen consumption exposed to the action of Bravo, Champion, Tilt and Tiradin fungicides in differrent concentrations are shown in fig. 5-8. Fig.2. The influence of Champion fungicide upon breathing frequency on prussian carp Fig.5. The influence of the Bravo fungicide upon oxygen consumption on prussian carp Fig.3. The influence of Tilt fungicide upon breathing frequency on prussian carp 85 Changes of some physiological parameters… / Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010) Clinical symptoms observed during fungicide exposure (Bravo, Champion, Tilt and Tiradin) of prussian carp, correspond to observations by other authors reporting on the toxicity of fungicides [12], [13], [14]. Common symptoms of initial acute exposure to fungicides have apparent fish hypoxia, disoriented (ataxic) at the surface, and mucus-producing effects. The oxygen consumption was found to be significantly influenced by the concentration of the used fungicides. Bravo 500 SC ,in concentrations of 0.78125 x 10-3, 1.5625 x 10-3, 3.125 x 10-3, 6.25 x 10-3 and 12,5 x 10-3 ml / l Bravo, had an overall stimulating effect on oxygen consumption of prussian carps in the first phase (with variable duration: 24-96 hours after exposure) followed by restoration of energy metabolism after 7 days of exposure to toxic. Tiradin and Tilt have an inhibitory effect on the energy metabolism of prussian carps. After 7 days of exposure to Tilt, for all lots of fish tested, oxygen consumption values fall below the value recorded before the introduction of fish in experiments. Decreased oxygen consumption under the action of some pesticides and changes in respiratory rate (Dithane M 45, Reldan, Tilt,) has also been noticed by Marinescu [12] and Ponepal [13], [14]. Figure 9 show the changes in the average values of erythrocytes after one and two weeks of exposure to some fungicides. Fig.6. The influence of the Champion fungicide upon oxygen consumption on prussian carp Fig.7. The influence of the Tilt fungicide upon oxygen consumption on prussian carp Fig. 9. The influence of some fungicide upon number of erythrocytes on prussian carp Fig.8. The influence of the Tiradin fungicide upon oxygen consumption on prussian carp 86 Maria Cristina Ponepal et al./ Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010) Champion After 7 and 14 days of exposure to three fungicide (Bravo, Champion and Tiradin) we found out a significant decrease in the number of erythrocytes. Similarly results were obtained in carp by Hughes [15] after a brief exposure to Methadathion. The decrease in RBC after 7 days exposure to some pesticides in fish was observed by Dhembare and Pondha [16], Ponepal et al. [13], [14]. The fungicide Tilt, in concentration of 1 ml/l water has an stimulatory effect of erythocytes number. In experimental variants with Tiradin and Tilt have only been observed three stages of the sympthomatologicycal scheme described by Schäperclaus for the intoxicated fish [10]. Neurotoxic effects in rats from thiram exposure has been noticed by Lee and Peters [17]. Table 1 shows the data on fish mortality during the experiments. Chlorothalonil toxicity is lower than that indicated in the literature [2], [3], which is due both to the testing method (semi-static) and the fact that no pure chemical product has been used. 0.03 0.3 3 Conc entrat ion ml/l, mg/l Bravo 0.000 7812 5 0.001 5625 0.003 125 0.006 25 0.012 25 Contr ol lot Contr ol lot 0.25 Tilt 0.5 1 Tiradin Table 1. Lethal effect of some fungicide on prussian carp Experimental variants (fungicide 0.003 2 4 Contr ol lot 0.01 0.02 0.04 0.08 0.16 Contr ol lot 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 8 7 1 0 10 10 10 10 8 10 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 6 4 1 0 10 10 10 10 7 10 1 0 1 0 1 0 9 9 9 9 9 8 8 9 8 7 8 6 4 1 0 1 0 1 0 1 0 3 0 1 0 10 10 10 8 6 10 1 0 1 0 1 0 9 10 9 10 10 9 9 9 8 2 0 1 0 10 10 10 8 4 10 0 0 10 0 0 9 10 10 8 6 1 10 10 10 6 5 1 10 4. Conclusions The number of living specimens Immersion time (hours) 24 48 72 96 168 10 10 10 10 10 33 6 10 10 10 10 10 9 9 10 10 9 9 8 7 10 10 9 9 8 6 10 10 9 8 7 2 10 10 10 10 10 10 The fungicides investigated (Bravo, Champion, Tilt and Tiradin) have changed the respiratory rhythm of prussian carps. For all concentrations tested the effect of Bravo and Tilt fungicide is initially stimulating and inhibitory as regards the frequency of respiratory movements. In two experimental variants (0.01 and 0.02 ml/l water) Tiradin is stimulating of the breathing frequency of fish; at the concentration of 0.04, 0.08 and 0.16 ml/l water, the fungicide caused a decrease in the respiratory rhythm of prussian carps. The fungicide Bravo, had an overall stimulating effect on oxygen consumption of prussian carps in the first phase followed by restoration of energy metabolism after 7 days of exposure to toxic. The fungicide Champion, under the concentrations of 0.003 and 3 mg/l water, had, after 87 Changes of some physiological parameters… / Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010) biochimia animală, Editura Didactică şi Pedagogică, Bucureşti, 252 pp. [12] MARINESCU AG, DRĂGHICI O, PONEPAL C, PĂUNESCU A, 2004 - The influence of fungicide (Dithane M-45) on some physiological indices in the prussian carp (Carassius auratus gibelio Bloch), International Association for Danube Research, Novi Sad, 35: 209-214 [13] PONEPAL MC, PĂUNESCU A, MARINESCU AG., DRĂGHICI O, 2009 - Effect of the Fungicide Chlorothalonil (Bravo) on Some Physiological Parameters in Prussian Carp, Lucrări ştiinţifice USAMV Iaşi, seria Horticultură, vol 52. [14] PONEPAL M., PĂUNESCU A, MARINESCU AG, DRĂGHICI O, 2009 - The Changes of Some Physiological Parameters in Prussian Carp Under The Action of the Tilt Fungicide, Bulletin UASVM, Cluj, 2009, 66. [15] HUGHES G., SZEGLETES T, NEMCSOK KJ. 1995 - Haematological and biological changes in the blood of carp (Cyprinus carpio) following brief exposure to an organophosphoric insecticide (Methidathion),Abs.Int.Biond.Symp.Cesze Budejovice, May [16] DHEMBARE AJ, PONDHA GM, 2000 Haematological changes in fish. Punctius sophore exposed to some insecticides, J.Expt. Zoo. India, 3(1), 41-44. [17] LEE CC and PETERS PJ, 1967 - Neurotoxicity and behaviour effects of thiuram in rats, . Envir. Health Perspectives, 17:35-43. 96 hours of exposure, a stimulatory effect on oxygen consumption for the prussian carp. The other two fungicides tested (Tiradin and Tilt) have an inhibitory effect on oxygen consumption for the prussian carps. After seven and 14 days of exposure to Bravo 500 SC (0.078125 x 10-3 to 12.5 x 10-3 ml/l water) and Champion (0,003 mg/l water) at 16-18 ºC we found out a significant decrease in the number of erythrocytes of prusian carp. Tilt 250, in concentration of 1 ml/l water causess a increase in the prussian carps erythrocytes (after 7 and 14 days of exposure). 5. References [1] http://extoxnet.orst.edu/pips/chloroth.htm [2] KIDD H and JAMES DR, 1991 - Eds. The Agrochemicals Handbook, Third Edition. Royal Society of Chemistry Information Services, Cambridge, UK, (as updated). 6-10 [3] DAVIES PE AND WHITE RWG, 1985 - The toxicology and metabolism of chlorothalonil in fish. 1. Lethal levels for Salmo gairdneri, Galaxias maculatus, G. truttaceus and G. auratus and the fate of super(14)C-TCIN in S. gairdneri , Aquatic Toxicology, 7 (1-2). pp. 93-105. [4] HORNE MT and DUNSON WA, 1995 - Effects of low pH, metals, and water hardness on larval Archives of Environmental amphibians, Contamination and Toxicology, 29:500-505 [5] THOMSON WT, 1997- Agricultural Chemicals. Book IV: Fungicides. 12th edition, Thomson Publications, Fresno, CA [6] http://www.epa.gov/ngispgm3/iris/irisdat [7] http://www3.bae.ncsu.edu/info1/courses [8] HOWARD PH, 1989 - Pesticides. In : Handbook of Environmental Fate and Exposure Data for Organic Chemicals, Lewis Publishers, Chelsea, MI, pp.4-20 [9] www.epa.gov/HPV/pubs/summaries [10] PICOS CA, NASTASESCU GH, 1988 - Lucrări practice de fiziologie animală. Tipografia Universităţii din Bucureşti, p.107, 122-123, 192195. [11] ŞERBAN M, CIMPEANU G, IONESCU EMANUELA, 1993 - Metode de laborator în 88 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 CYTOGENETIC EFFECTS INDUCED BY MANGANESE AND LEAD MICROELEMENTS ON GERMINATION AT TRITICUM AESTIVUM L. Elena DOROFTEI1, Maria Mihaela ANTOFIE2, Daciana SAVA1, Marioara TRANDAFIRESCU1 1 Faculty of Natural and Agricultural Science, „Ovidius” University, Constantza, University Street No. 1, Bilding B, Campus, 900552, Romania, email: edoroftei2000@yahoo.ca 2 Faculty of Agricultural Sciences, Food Industry and Nature Potection, University “Lucian Blaga”from Sibiu __________________________________________________________________________________________ Abstract: Our study is about the effects of manganese and lead microelements treatment on germination at Triticum aestivum L. The cytogenetic effects were studied by the calculation of the mitotic index, by the study of the interphase and chromosomal aberrations on the mitotic cells. We used MnSO 4 and Pb(NO 3 ) 2 solutions with different concentrations: 0.0001, 0.005, and 0.01%. The Triticum seeds were preliminary imbued in water, and then they were treated for 6 and 24 hours in these solutions. The control group was treated with water. We prepared five cytological slides, for each slide we have studied 10 microscopic fields with good density of cells for the mitotic index and another 10 different microscopic fields for abnormal interphases and chromosomal aberrations. In the analyzed meristematic cells we observed an almost totally inhibition of cell division and the mitotic index was smaller in comparison with the control variant. The study of the frequency of the cells in different phases of the mitotic division showed that the highest percent was registered by prophases, followed at distance by telophases. We can conclude that the heavy metals Mn and Pb have a significant mutagenic activity in vivo upon the radicles of Triticum aestivum L. Keywords: Triticum aestivum, cytogenetic effects, lead, manganese, mitotic index, chromosomal aberrations. __________________________________________________________________________________________ 1. Introduction Aside pesticides - the most important „stress indicators” which are especially used in agriculture, other very important indicators are heavy metals. The residual waters resulting from the galvanic industry contain a real “hurricane” of heavy metals such as: mercury, cadmium, zinc, copper, lead and chrome. Generally the water pollution sources for heavy metals are as following: galvanic industry, mining, metallurgy and car industry. Copper water pollution is especially due to viticulture as the copper sulphate is used for pests’ control. Lead is eliminated mostly as a result of burning gasoline, petrol and different dyes, affecting the central nervous system in humans, creating behaviour problems and convulsions, at higher levels being lethal. Lead is spearing no organ or system being the first incriminated in boosting or getting worse a series of diseases through diminishing the body resistance. Lead effects are usually irreversible. ISSN-1453-1267 Manganese is a nutritionally essential chemical element but also in certain conditions it can be potentially toxic. Manganese name is originating from Greek language meaning “magic” and this feature is still adequate because the scientists are still working to understand different effects of its deficiency and toxicity effects for living organisms. However, without doubt in high levels manganese is highly toxic causing a series of pathologies based on reactive oxygen species (ROS) generation. Long term oxidative stress consequences in human where associated to the different diseases pathogenesis and toxicities namely atherosclerosis, diabetes, chronically inflammatory diseases, neurological disturbances and cardiovascudiseases. Manganese induces the oxidative stress in a time and concentration depending manner, according to the cytotoxic parameters measurements, lactate dehydrogenase and lipid peroxidation. Also, manganese may accumulate into the cell causing cytotoxic effects and cell © 2010 Ovidius University Press Cytogenetic effects induced by manganese... / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) destruction. Following different activity enzyme alteration and the alteration of gene expression the intracellular disruptions caused by manganese include DNA helix broken up, chromosomes destruction and lipid peroxidation (Brooks, 1994). Our research focused in detecting the mutagenic effects induced by heavy metals such as manganese (Mn) and lead (Pb) on higher plants using the cytogenetic analysis in Triticum aestivum L. as plant indicator for heavy metals polluting degree in crops. The toxicity symptoms induced by heavy metals in plants are the results of some negative effects on physiological processes including: respiration and photosynthesis inhibition, water – plant relationship disruption, decreasing plasmalema permeability in root cells, adverse effects on the metabolic enzymes (Arduini, 1994; Chardonneres et al., 1999; Ouzounidou, 1994; Vangronsveld and Clijsters, 1994; Vennitt and Parry, 1984). Novex Holland digit camera was used for taking photographs. Table 1. Heavy metal concentrations and durations used for Triticum aestivum seeds treatments Heavy metal MnSO 4 MnSO 4 Pb(NO 3 ) 2 Pb(NO 3 ) 2 0,0001% 0,005% 0,01% Variant name V1 V2 V3 Treatment duration 6 hours 0,0001% 0,005% 0,01% 0,0001% 0,005% 0,01% 0,0001% 0,005% 0,01% V4 V5 V6 V7 V8 V9 V10 V11 V12 24 hours Concentration 6 hours 24 hours In this study 5 slides per variant were analyzed and for each slide 10 microscopically filed were used for mitotix index calculation and for chromosomal aberrations study. 2. Materials and Methods The chemical effects on chromosomes are often studied on plant material such as root tips as they are easily produced through seed germination, the experiments may be conducted all over the year and are not costly (Bateman,1977). For studying the heavy metal effect on mitosis we used solutions of MnSO 4 and Pb(NO 3 ) 2 in different concentrations (0,0001%; 0,005% and 0,01%) in which were submersed Triticum seeds for 6 and 24 hours, in Petri disches. As control it was used tap water. Fragments of young roots were fixed into a mixture solution of ethylic alcohol and glacial acetic acid in a volumetric rapport of 3:1 for 16 h in refrigerator followed by a gentle acidic hydrolysis in HCl 1N solution for 5 min at 60°C. The roots are coloured through the Feulgen method using the Schiff reactive for 90 min followed by a water bath for 20 min. The slides were prepared applying squashing method and the samples were analyzed in light microscopy for the cytogenetic effects of heavy metals by calculating the mitotic index and revealing the chromosomal aberrations for different mitotic stages (Doroftei et al., 2008). A 3. Results and Discussions Analyzing the control untreated roots it was revealed the normal feature of the chromosomes and also normal cell division behaviour with a mitotic index of 15 %. Roots development was lower when the Triticum seeds were immersed into the tested solutions, macroscopically differences being observed compared to the control. Thus, the treated roots were smaller and in a low number compared to the control. The mitotic index significantly decreased especially in the case of 0.01% and 24 h treatment duration for manganese and lead too, supporting the idea that cell division is slower progressing compared to the control (Tab. 2). These microscopically observations are supported by those macroscopically (root number and size). The chromosomal aberrations are relatively diverse, being aleatory distributed and depending 90 Elena Doroftei et al. / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) on manganese and lead concentration and treatment period. For a treatment with MnSO 4 solution in concentration of 0.01% for 24 h treatment, it was observed cells with big nuclei and unorganized and vacuolated features. The un-organizing process is due probably to some disequilibrium occurred as a consequence of genetic material accumulation in a too big quantity. The treatment with MnSO 4 solution in concentration of 0.01% for 6 h treatment it was observed that the majority of cells were in interphase and prophase and after 24 h of treatment cell plasmolysis occurred for non dividing cells. The treatment with Pb(NO 3 ) 2 solution in concentration of 0,0001% and 0,005%, for 6 or 24 h treatments, induced a decrease in mitotic division frequency. For a treatment with Pb(NO 3 ) 2 of 0,01%, for 24 h a significant decrease cells in mitotic division frequency it was registered as a result of summing the effects of high concentration and long period of treatment. For this later variant, nuclei unregulated in shape and size were observed and chromosome appeared either big with a relaxed chromatin either small but presenting a compact chromatin and unregulated shape. For lead too, for a concentration of 0.01% for 6 h there were observed predominantly cells in interphase or prophase and after 24 h of treatment the cell plasmolysis occurred in the non-dividing cells. The studied heavy metals solutions may have according to our results the following negative effects: 1. slowing down the cell division rate (figs. 1-16) 2. frequent cell degradation appearance (figs. 7,8,9,10,11,13,14 ) 3. dehydration effect at cell level frequently inducing cell plasmolysis, more drastically at 24 h treatment (figs. 9,10,11,15,16,17) 4. heterochromatinization during prophase (figs. 6,7,8,13,14) 5. changes in the nuclei shape becoming elongated (figs. 6,7,8) degradation of the nucleic material in the completed destroyed cells (figs. 6,7,8,13,14) 7. an early chloroplasts formatting can be observed (figs. 9,10,12,15,16,17). In all variants, comparing to the control, a decreasing in the mitotic index was observed (figs. 1, 2, 3, 4). We recorded the lack of cells in anaphase for the following variants: 3, 6 and 11. For the control as well as for the treated variants the predominance of prophase and telophases towards the metaphases and anaphases was registered (fig. 5). The highest percentage of dividing cells is registered for the variant no. 1 (4%) and it is shown in tab. no. 2. The biggest number of cells in prophase (19) in variant 1 was registered (tab. 2). For metaphase the biggest number of cells was registered in variant 7 (14) followed by in variants 1 and 9 (11). These data support the idea that among heavy metals, manganese in large quantities impedes the normal roots growth for higher plants as a consequence of cell division negative effects induced for the meristematic cells in Triticum aestivum. Faze de diviziune % 6. 6 5 4 3 2 1 0 Profaze Metafaze Anafaze Telofaze Fig. 1. Mitotic division phase’s frequency in Triticum aestivum treated with MnSO 4 for 6 hours. 91 Faze de diviziune % Cytogenetic effects induced by manganese... / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) 6 5 4 3 2 1 0 Profaze Metafaze Anafaze Telofaze Faze de diviziune % Fig. 2. Mitotic division phase’s frequency in Triticum aestivum treated with MnSO 4 for 24 hours. Fig.5. Root meristematic cells in control of Triticum aestivum. It can be observed cells in prophase, metaphase, anaphase and telophase and cytochinesis (200x). 6 5 4 3 2 1 0 Profaze Metafaze Anafaze Telofaze Faze de diviziune % Fig.3. Mitotic division phase’s frequency in Triticum aestivum treated with Pb(NO 3 ) 2 for 6 hours. 6 5 4 3 2 1 0 Profaze Metafaze Anafaze Telofaze M t V i t V i t Fig.6. Root meristematic cells in Triticum aestivum treated with MnSO 4 0.0001% for 6 h. It can be observed cells without content and cells in prophase and telophase. The nuclei are hypertrophic with obviously hetero-chromatinisations and vacuolisations (150x). V i t Fig.4. Mitotic division phase’s frequency in Triticum aestivum treated with Pb(NO 3 ) 2 for 24 hours. 92 Elena Doroftei et al. / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) Fig.7. Root meristematic cells in Triticum aestivum treated with MnSO 4 0.0001% for 6 h. It can be observed cells without content and cells in prophase and cytochinesis. The nuclei are hypertrophic with obviously hetero-chromatinisations and vacuolisations (150x). Fig.9. Root meristematic cells in Triticum aestivum treated with MnSO 4 0.01% for 6 h. It can be observed abnormal disposed cells without content mixed with plasmolytic cells in witch we can observed an early chloroplasts formatting (600X). Fig.8. Root meristematic cells in Triticum aestivum treated with MnSO 4 0.005% for 6 h. It can be observed plasmolytic cells mixed with cells in prophase and telophase abnormal disposed and containing hypertrophic nuclei with obvious heterochromatinisations (150X). Fig.10. Root meristematic cells in Triticum aestivum treated with MnSO 4 0.0001% for 24 h. It can be observed long cells disposed in lines; cells are plasmolytic, during prophase and in witch we can observed an early chloroplasts formatting (600X). 93 Cytogenetic effects induced by manganese... / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) Fig.11. Root meristematic cells in Triticum aestivum treated with MnSO 4 0.005 % for 24 h. It can be observed long cells disposed in lines; cells are plasmolytic, during prophase (600X). Fig.13. Root meristematic cells in Triticum aestivum treated with Pb(NO 3 ) 2 0.0001 % for 6 h. It can be observed cells without content alternating with cells in prophase and telophase, having nuclei hypertrophic (400X). Fig.12. Root meristematic cells in Triticum aestivum treated with MnSO 4 0.01% for 24 h. It can be observed long cells disposed in lines; cells are strongly plasmolytic, during prophase and in witch we can observed an early chloroplasts formatting (600X). Fig.14. Root meristematic cells in Triticum aestivum treated with Pb(NO 3 ) 2 0.005 % for 6 h. It can be observed cells without content alternating with cells in prophase and telophase, having nuclei hypertrophic (400X). 94 Elena Doroftei et al. / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) Fig.15. Root meristematic cells in Triticum aestivum treated with Pb(NO 3 ) 2 0.01 % for 6 h. It can be observed long cells disposed in lines; cells are plasmolytic, during prophase and in witch we can observed an early chloroplasts formatting (600X). Fig.17. Root meristematic cells in Triticum aestivum treated with Pb(NO 3 ) 2 0.01 % for 24 h. It can be observed long cells disposed in lines; cells are plasmolytic, during prophase and in witch we can observed an early chloroplasts formatting and curly cell’ s walls (600X). 4. Conclusions Based on the results of this study we may conclude that: The heavy metals solutions used in this experiment have a great mutagenic effect on the root meristematic cells of Triticum aestivum After the heavy metals solution treatment a decrease in cell division in rate was recorded The heavy metals have a dehydration effect at cellular level In all variants a decrease in the mitotic index compared to the control was observed The mutagenic effects depends on the used heavy metals in the treatment and the treatment duration In the treated cells an early chloroplasts formatting can be observed. Cytogenetic tests on Triticum aestivum reveal a decrease in mitotic index after the treatment with the heavy metals solutions. These results revealed that the studied heavy metals present a significant Fig.16. Root meristematic cells in Triticum aestivum treated with Pb(NO 3 ) 2 0.005 % for 24 h. It can be observed long cells disposed in lines; cells are plasmolytic, during prophase and in witch we can observed an early chloroplasts formatting and curly cell’ s walls (600X). 95 Cytogenetic effects induced by manganese... / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) mutagenic activity. The inhibition of mitotic division in the root apex induces the root growth tolerant and sensitive Silene vulgaris. J. Plant. Physiol., 155(6), 778-787. [4] OUZOUNIDOU, G., 1994: Cooper induces changes on growth, metal content and photosynthetic function of Alysum montaneum L. plants, Environ. Exp. Bot., 34, (2), 165-172. [5] VANGRONSVELD, J., CLIJSTERS, H., 1994: Toxic effects of metals. In: Plants and the chemical elements, 150-177. Edited by M.E. Farago. Ed. VCH, Weinheim, New York, Basel, Cambridge, Tokio. [6] VENNITT, S., PARRY, J.M., 1984: Mutagenicity testing: a practical approach. Ed. IRL Press, Oxford, Washington DC. [7] BATEMAN, A.J., 1977: Handbook of mutagenicity - Test procedures. Edited by B.J. Kilbey, M. Legator, W. Nichols, C. Ramel. Ed. Elsevier, North Holland, Amsterdam. [8] DOROFTEI, E., MIRON, L., ROTARUSTĂNCIC, M., 2008: Efectul mutagen al metalelor grele cupru şi cadmiu la Allium cepa L. (The mutagenic effect of heavy metals cooper and cadmium at Allium cepa L.) În: Ardelean, A., Crăciun, C. (eds), Analele Societãţii Naţionale de Biologie Celularã, XIII, 225-229, Risoprint, Cluj-Napoca. inhibition as an active reaction of the plant when plants are exposed to the action of heavy metals in soil. Heavy metal effects are more profound but they may become visible using further molecular techniques. These results are sufficient serious arguments in the elaboration of prophylactic methods for pollution combating of surface land water, underground water as well as for grounding the protection measures for ecosystem maintaining. 5. References [1] BROOKS, R.R., 1994: Plants and hyperaccumulate heavy metals. In: Plant and chemical elements, 88-105. Edited by M.E. Farago. Ed. VCH, Weinheim, New York, Basel, Cambridge, Tokio. [2] ARDUINI, I., GOLDBOLD, D.L., ONNIS, A., 1994: Cadmium and cooper change root growth and morphology at Pinus pinea and Pinus pinaster seedling. Physiol. Plant., 92, 675-680. [3] CHARDONNERES, A.N., BOOKUM, W.M., VELLINGE, S., SCHAT, H., VERKLEIJ, J.A.C., ERNST, W.H.O., 1999: Allocation patterns of zinc and cadmium in heavy metal 96 Elena Doroftei et al. / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) Table 2. Number of analysed cells for citogenetic studies regarding the effects of heavy metals manganese and lead on cell division Variant Total studied cell Nr. Total interphase cells Nr. % Total division cells Nr. % Total prophase cells Nr. % Total metaphase cells Nr. % Total anaphase cells Nr. % Total telophase cells Nr. % Martor 1000 850 85 150 15 51 5,1 40 4,0 30 3,0 29 2,9 V1 1000 960 96 40 4,0 19 1,9 11 1,1 6 0,6 4 0,4 V2 1000 968 96,8 32 3,2 14 1,4 6 0,6 4 0,4 8 0,8 V3 1000 981 98,1 19 1,9 9 0,9 4 0,4 - - 6 0,6 V4 1000 980 98 20 2,0 10 1,0 5 0,5 4 0,4 1 0,1 V5 1000 976 97,6 24 2,4 11 1,1 6 0,6 3 0,3 4 0,4 V6 1000 975 97,5 25 2,5 12 1,2 9 0,9 - - 4 0,4 V7 1000 966 96,6 34 3,4 9 0,9 14 1,4 5 0,5 6 0,6 V8 1000 965 96,5 35 3,5 15 1,5 9 0,9 6 0,6 5 0,5 V9 1000 966 96,6 34 3,4 12 1,2 11 1,1 8 0,8 3 0,3 V10 1000 974 97,4 26 2,6 11 1,1 4 0,4 3 0,3 8 0,8 V11 1000 979 97,9 2,1 2,1 12 1,2 6 0,6 - - 3 0,3 V12 1000 979 97,9 2,1 2,1 11 1,1 4 0,4 1 0,1 5 0,5 97 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 PROBLEMS OF THE HARMONIZING ENVIRONMENTAL LEGISLATION AT THE COMPARTMENT "PISCES" IN THE REPUBLIC OF MOLDOVA Petru COCIRTA, Olesea GLIGA Institute of Ecology and Geography (Academy of Sciences of Moldova). Academy Str. No.1, Chișinău, MD-2028, Republica Moldova E-mail: pcocirta@hotmail.com, camiprim@inbox.ru __________________________________________________________________________________________ Abstract: In the paper are presented some results regarding principal characteristics on the structure, qualitative and comparative analysis of the national acts with EU directives as well with EU and ISO standards. It was demonstrated the compatibility of some national legislation and normative acts with EU ones. Special attention was dedicated to the rare and endangered species of fish. It was created databases on environmental legislativenormative acts of the Republic of Moldova at the compartment “Fishes”, which shows a various and satisfactory number of acts in this domain. In the final part of the paper are presented some conclusions and proposals on the development of legislation and norms regarding fish species in the Republic of Moldova in accordance with EU and international requirements. Keywords: fishes and environmental legislation and normative acts state. __________________________________________________________________________________________ 1. Introduction According to the Declaration of Rio de Janeiro in 1992 and Agenda 21 [1], protection of biological diversity is one of the global environmental problems, which depends on addressing the quality of life and existence of the living organism son the earth. Development and conservation of the diversity of ichthyofauna species are of paramount importance in the management of biological diversity in the marsh and aquatic ecosystems in the Republic of Moldova [2]. In the past 100 years anthropogenic pressure on aquatic and march ecosystems has changed cardinal the quantity and quality of aquatic biological diversity. In Republic of Moldova the aquatic and marsh (water areas of rivers, lakes, dam lakes, ponds) ecosystems were limited to 94,6 thousand ha (2.8% of total territory), and are unevenly distributed and characterized by a wide variety of ecological, physical, geographical, hydrochemical, hydrobiological etc. particularities. Hydrographical ISSN-1453-1267 network consists of three main rivers - the Danube, Dniester and Prut, as well as of 3260 rivulets and 3532 lakes. Most of rives were damaged, destroyed or channeled. Biodiversity includes 160 flora and 125 fauna (vertebrates) species. Hydrofauna recorded over 2135 species, including ichthyofauna - 82 species [3-5]. In recent decades the influence of anthropogenic factors (industrial pollution, eutrophication progressive, toxicity, reducing water flow, etc.) upon ecosystems river Dniester and Prut, small rivers in the territory of the country makes major changes in biodiversity of the hydro-biocenozes with loses the viability and biological significance of rivers into the biosphere and environment. As a consequence, fish resources, which are one of the important indicators statuses of the aquatic ecosystems, decreased sharply in majority natural water objects of the Republic of Moldova and a number of species (sterlet, barbel, zarte and others) are endangered. In the Red Book of the Republic of Moldova (Second edition, 2001) [6] it was included © 2010 Ovidius University Press Problems of the harmonizing environmental… / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010) 12 species of fishes (14,6 % of total number). Acording to investigations made by Usatai [7], it ensuring of the environmental management in biodiversity conservation domain. In this work are presented analytical information on current level of legislative-normative takes place the process of replacement of valuable species with less valuable. Political and socio-economic reforms in Moldova ware conditioning the need to change of attitudes towards use of natural resources, promoting economic and social development compatible with the environment. After the 2009 parliamentary elections was conditioned the need to promote the new ideas and actions for to harmonize relations in the system “Man-Society-Nature”. In this context in the Republic of Moldova there are implemented the National Strategy „Agenda 21” [8] and a number of existing national programs: The Moldovan Village, 2005-2015; Program of the stabilization and economic recovery of the Republic of Moldova for the years 2009-2011 [5], and new one: Rethink Moldova. Priorities for Medium Term Development, 2010-2013 [10], which would help “de facto” to economic development through solving the environmental problems and respectively to stop the pollution of the environmental and degradation their components. Current state of water areas of the Republic of Moldova induces new provocations on the elaboration of measures to develop the actions and current species diversity of ichthyofauna, the improvement, utilization and sustainable conservation of hydrofauna in general. In the program „Rethink Moldova. Priorities Medium Term Development” among other priority issues that require to be solving there are the approximation of legislation and normative acts to those of the European Union. Achieving these desiderates requires needs updating of the existing legislative-normative base, elaboration new laws and regulations and/or modification of those existing, adaptation national standards and normative to those international ones and/or takeover of international standards of the ISO and EN Series, etc. This desiderates will fully covers the domain “Ichthyofauna”, including the section “Fishery”. A comprehensive study should be carried out in full for the evaluation of legal-normative basis and for highlighting some perspective problems for to legal assurance of the environmental management of ichthyofauna species diversity in the Republic of Moldova and on forming of the base of legislativenormative acts in this domain. The aim of work: - analyze of legal and normativ systems on compartment "Fishes" of UN, EU and Republic of Moldova; - assessment and completing the data base of the acts referred to the Republic of Moldova; Highlighting the problems of the legislativenormative development in the Republic of Moldova on ichthyofauna domain; - elaboration of the proposals for harmonization of legislation and normative in the domain of ichthyofauna to the Strategy of Sustainable Development of the Republic of Moldova, to the respective EU and international acts; In this work are presented analytical and summary information on the current level of insurance protection activities of ichthyofauna in the Republic of Moldova and creation of the base of legislative-normative acts in referred domain. 2. Material and Methods Study of information regarding legislativenormative acts was performed through analyse of the data banks, catalogs and other official materials of the international and national environmental organizations. Collecting of acts materials was effectuated in the frame of the official publications (written or electronic forms) of Secretariats of the international conventions, International Organization for Standardization, European Union and others, as well as from the Republic of Moldova - periodical publication "Monitorul Oficial a Republicii Moldova", Websites of the Parliament, Government, Ministry of Justice and Ministry of Environment and others. Given the fact that information accumulated in ihthiofauna domain will serve to comparative analysis of national acts to those international, especially to 100 Petru Cocirta, Olisea Gliga / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010) European ones and will be used to develop concrete recommendations on the harmonization of legislation, Box 1: Multilateral environmental agreements on nature protection normative and standards, it was take into account the • Convention on Wetlands of International respective international methodological Importance Especially as Waterfowl Habitat (Ramsar, 1971) recommendations [11-18] and the those of national • Convention on International Trade in order - Standards of the Republic of Moldova on the Endangered Species of Wild Fauna and principles and methodology of standardization (SM Flora (Washington, 1-0:2003, MS 1-12:2002; SM 1-20:2002, SM 1• 1973) 21:2002 [18, 19]) and others. • Convention on Conservation of Migratory Storage of the specialized information and Species of Wild Animals (Bonn, 1979) creation of databases of legislative-normative acts on • Convention on the Conservation of biodiversity domein (EU Directives, International European Wildlife and Natural Habitats Conventions, National legislation and normative) was (Bern, 1979) made in electronic form. • Convention on Biological Diversity (Rio de Collecting and processing of information on Janeiro, 1992) standards and technical regulations was effectuated by • Convention on Cooperation for the using existing databases of international and national Protection and Sustainable Use of the Websites and formation of a register of operative Danube River (Sofia, 1994) information in this domain. 3.1.2. EU legislation In accordance with EU recommendations [1014], were taken to record the majority of legislative and normative acts, which are part of the acquis of Environment and need to be transposed into national law. Environmental acquis recommended for harmonization of national legislation is considerably smaller (118 documents). As the compartment “Fish” there was highlighted the following: EU Fish Protection Legislation. Within this framework, EU Nature conservation policy is implemented by one main piece of legislation – Habitats Directive - the Council Directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora. The Directive aim to provide protection for listed species and habitats and to create the European ‘coherent European ecological network of sites – called Natura 2000 to enable the maintenance or restoration of natural habitat types and the habitats of species at favorable conservation status (Art. 3, Habitats Directive). The Habitats Directive requires Special Areas of Conservation (SACs) to be designated for listed plant and animal species, and habitats. Together, SACs and Special Protection Areas (SPAs) from Birds Directive (Council Directive 79/409/EEC of 2 April 1979 on the conservation of wild birds) make up the Natura 2000 sites. SPAs and 3. Results and Discussions In the Program "Rethink Moldova. Medium Term Development Priorities, 2010-2013” approximation of legislative and normative acts to those of the EU is among the priority issues, that need resolving operational. Collection and analysis of material under mentioned program has permitted to highlight the following aspects of the assessment and the need of International, European and National acts for the ecological management on "Fish" compartment in the Republic of Moldova. 3.1. Assessment and training normative legislative base in "PISCES" 3.1.1. International legislation Various multilateral environmental agreements or conventions have been concluded for nature protection in general, and for aquatic fauna in special. The European Community takes an active part in the elaboration, ratification and implementation of multilateral environmental agreements. Republic of Moldova also is a part of those conventions and made different action in accordance with ratified conventions. The principal of them which cover also the Fish compartment are named chronologically below (see Box 1). 101 Problems of the harmonizing environmental… / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010) SACs areas can overlap. The Natura 2000 network already comprises more than 20,000 sites, covering almost a fifth of the EU territory. and normative in the ichthyofauna domain is satisfactory. Political and social reforms from recent years have highlighted the need to harmonize legislativenormative acts, inclusive the ichthyofauna domain, to Besides this directive there are further relevant pieces of EU nature protection legislation referred to fish, summarized in Box 2. the international requirements, which will allow fulfilling the obligations of the Republic of Moldova Government, assumed by signing the international environmental conventions and facilitating the process of integration in European Union. In this context, there is evident the tendency and efforts for significant changes of legislative-normative acts of the Republic of Moldova, which started in last 5-6 years by applying the mechanism of their harmonizing to requirements of the international legislation and normative, and, in particular, to the European requirements, in accordance with international obligations of the country. However, we should mention that several legislative-normative acts from Republic of Moldova have prescriptive nature and contain general provisions that regulate, primarily, the relations of animal kingdom protection and conservation, the management of the state protected natural areas and others. There is poorly developed legislative base for protection of natural complexes, for creating a green housing (frame) and application of stringent measures for recovery of environmental condition, which have directly impacts the habitats of fish species, a special vulnerable species. In national legislation lacks the mechanism needed to optimal ensuring of the protection and conservation activities of natural habitats of many species of fish, as well as of communities of the aquatic plant and animals. Box 2: EU nature (fish) protection related legislation • Council Directive 92/43/EEC on the conservation of natural habitats and of wild fauna and flora • Council Directive 1999/22/EC relating to the keeping of wild animals in zoos • Council Regulation (EC) No. 338/97 on the protection of species of wild fauna and flora by regulating trade therein Other EU legislation relevant to nature (fish) protection include: • Environmental Impact Assessment Directive (85/337/EEC), amended by Council Directive 97/11/EC, • Access to Environmental Information Directive (90/313/EEC), • Reporting Directive (91/692/EEC). 3.1.3 Legislative-normative acts of the Republic of Moldova. On June 1, 2010 database of legislativenormative acts of Republic of Moldova in the ichthyofauna domain and interdependent ones represents an impressive set of legal materials, namely: • 6 international environmental conventions to which Moldova is party; • 13 Laws of the Republic of Moldova; • 1 Presidential Decree of Republic of Moldova; • 44 acts of the Republic Moldova subordinate to laws, from wich 7 Decisions of Parliament, 35 Decisions of Government, 2 Acts of the Central Environmental Authority; • 1 Concept; • 2 Strategies; • 2 State Programs. The above mentioned has highlighted the importance of databases in this domain and the need to maintain and develop them. Analyse of the results obtained show that the development of legislations 3.1.4 Legislative issues on the conservation of rare and vulnerable species Republic of Moldova legislation covers the most part of the activities of rational use and conservation of ichthyofauna species (Law on Animal kingdom (1995), Law on State Protected Areas Fund (1998), Law on fund of fisheries, fisheries and fish culture (2006), Red book of Moldova (2001). Special attention is devoted to rare and vulnerable fish species that are protected by several legislative acts, the main ones being: the Law on State Protected Areas Fund, which includes 15 102 Petru Cocirta, Olisea Gliga / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010) species, the Red Book of Moldova - 12 species. We CD, BC also note the primary importance of the Berne 11.Gobio albipinnatus Convention (1979) to which Moldova is part from the (Vladykov Fang) – White-finned year 1993. Gudgeon CD, BC 12.Rhodeus sericeus amarus (Bloch) – European Bitterling Harmonize national legislation with Order Gadiformes international and European requirements impose LAK, LPA, RB, 13.Lota lota (L) - Barbot ERL additional measures to conserve species of Order Perciformes ichthyofauna. Were subjected to comparative analysis LAK, LPA, RB, 14.Zingel zingel (L) – Zingel some legislative acts of Republic of Moldova and ERL, CD, BC those more important international (European Red LAK, LPA, RB, 15.Zingel streber (Siebold) List (2009) [21], Council Directive 92/43/EEC [22] ERL, CD, BC Sreber and the Berne Convention [23] regarding the status Order Acipenseriformes and the protection state of rare and vulnerable fish 16.Gimnocephalus schraetzer species. It were analyzed the status of 21 important CD, BC (L) - Schraetzer species of fish presents on territory of the Republic of LAK, LPA, RB, 17.Huso huso (L) Moldova (Table 1). ERL, CD, BC European Sturgeon LAK, LPA, RB, CD 18.Acipenser guldenstaedti colchilus (V.Marti) – Russian Table 1. Some important fish species of the Republic Sturgeon of Moldova under comparative analysis 19.Acipenser stellatus (Pallas) - LAK, LPA, RB, CD, BC Sturry Sturgeon Name of Species Acts with LPA, CD, BC 20.Acipenster ruthenus (L) species found Sterlet Order Salmoniformes CD 21.Acipenster nudiventris LAK, LPA, RB, 1. Hucho-hucho (L) – Danube 1) (Lovetyki) – Bastard Sturgeon ERL, CD, BC salmon or Huchen 1) Note: LAK - Law on Animal Kingdom, LPA - Law on State Protected Areas Fund, RB - Red book of Moldova , ERL - European Red List, CD - Council Drective 92/43/EEC, BC - Bern Convention. 2. Salmo salar (L) ERL, CD, BC Atlantic salmon LAK, LPA, RB, BC 3. Umbra krameri(Walbaum) – European Mudminnow Order Cipriniformes LAK, LPA, RB, 4. Rutilus frisii Nordmann – ERL, CD, BC Black Sea Roach LAK, LPA, ERL 5. Leuciscus leuciscus (L) – Common Dace LAK, LPA, RB, 6. Leuciscus idus (L) ERL, BC - Ide or Golden Orfe LPA, ERL 7. Vimba-vimba (L) – Zarte 8. Barbus barbus borysthenicus LAK, LPA, RB, ERL (Dubowsky) – Borys 9. Barbus meridionalis (Petenyi LAK, LPA, RB, Heckel) – Mediterranien Barbel ERL, CD, BC 10.Cobitis taenia (L) – Spined Loach Analysis demonstrates that of these above mentioned, only six species (Danube Salmon, Black Sea Roach, Mediterranean Barbel, Zingel, Streber, European Sturgeon) are covered by all legislation acts under review, 12 species are covered by the European Red List, 15 species - Council Directive 92/43/EEC and 15 species - Bern Convention, respectively. There were identified 9 species, which in accordance with requirements of Council Drective 92/43/EEC falling under Annex II and requires the designation of special areas of conservation, as wel as under the Bern Convention, ratified by the Moldovan Parliament decision No. 1546-XII of 23. 06. 93. But 5 of these species (Salmo salar, Cobitis taenia, Gobio albipinnatus, Rhodeus sericeus amarus, CD, BC 103 Problems of the harmonizing environmental… / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010) Gimnocephalus schraetser) have no-one protected status in the Republic of Moldova. Also were identified 2 species (Acipenster rutenus and Acipenster nudiventri) falling under Annex V of the Council Directive 92/43/EEC, but the second species has no one of any protected status in country. The comparative analysis demonstrates the need to review the rarity status of the mentioned fish for section "Fish" can be found 17 ISO standards and one in elaboration and for section "Fishing and fish breeding" another 13 ISO standards. Meanwhile at the European level [18] for the section "Fish" it was highlighted 12 EN standards, from which 8 ISO standards taken by EN (European Normatives) organization. species and/or their inclusion in the Red Book of Moldova, in other legislation acts and/or performing other actions to perpetuate their best. These findings are in full compliance with existing legal basis of the Republic of Moldova: Art. 9, 16, 17 and 18 of the Law on the Red Book of Moldova (No. 325-XVI from 15. 12. 2005); The Common Action Plan Republic of Moldova – European Union, 2005-2007; and The Program "Rethink of Moldova. Priorities for medium term development". Comparative analysis of the state and how to protect rare and vulnerable species of fish confirmed the importance of measures taken and existing needs in Moldova in this section. Increasing vulnerability a ichthyofauna species confirmed by increasing number of introduced species in the Red Book of Moldova, the second edition of. Proposals have already been developed [7] introducing other four species in the next edition (III) of the Red Book. They are supposed to be: Tench - Tinca tinca (L), Spirlin - Alburnoides bipunctatus rossicus (Berg), Crucian Carp Carassius Carassius (L.), Wels catfish - Silurus glanis (L.). 3.2.2. Standards of the Republic of Moldova The Republic of Moldova doesn’t have in action ISO and EN standards. But in Catalogue of normative documents in standardization of the Republic of Moldova [19.20] in 65.150 section “Fishing and fish farming” were not identified standards in use, and in section 67.120.30 “Fish and fish products” are in force 109 GOST standards (standards of Russia adopted as national). It is clear the need of some decisions and activities in developing regulations and standards in the relevant field. 4. Conclusions Republic of Moldova dispose a significant base of legislative-normative acts in ichthyofauna domain. The legal regulation of ichthyofauna conservation is in continuous development, already having a solid theoretical and practical basis. In the last 4-5 years it is obvious trend and efforts for significant changing of the legislativenormative acts of the Republic of Moldova by applying of the mechanism to their harmonizing to the requirements of international legislation and normative, especially, to the European requirements. In connection with the increasing vulnerability of species further efforts are needed for continuous development of legislative basis regarding the habitats protection of vulnerable species of fish and protection of natural complexes in general, as well as creation a ecological housing and application of stringent measures to redress the environmental status. Comparative analysis of the state and protection mode of rare and vulnerable fish species has confirmed the importance of measures taken and existing needs in the Republic of Moldova in this domain. Additional measures are needed on improving the mechanism and instruments to ensure optimal 3.2. Evaluation and formation standards database in „PISCES” In the compartment "Pisces", relative to other domains there are few sets of standards, gained worldwide by international organizations - ISO (International Standardisation Organisation) and IEC (International Electrotechnical Commission) and at the European level - CEN (European Committee for Standardization), CENELEC (European Electrotechnical Committee for Standardization) and ETSI (European Telecommunications Standards Institute) [17.18]. 3.2.1. International standards In accordance with electronic information provided by the Organization ISO [17], in querying 104 Petru Cocirta, Olisea Gliga / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010) operation of the protection and conservation of many [9] National program “Moldovian Village, 2005species of fish natural habitats, as well as of plant and 2015; Program for stabilization and re-launch of animal aquatic communities. the economy in the Republic of Moldova for years In Republic of Moldova the ichthyofauna 2009-2011 (In Romanian) - www.gov.md. [10] Government of Moldova. Rethink Moldova. domain needs to move quickly to adopt international Priorities for Medium Term Development. Report and European standards in national practice. for the Consultative Group Meeting in Brussels Given that the diversity of fish species in 24 March 2010 Moldova is in its own way, unique and, under current http://siteresources.worldbank.org/INTMOLDOV A/Resources/Rethink-Moldova-2010-2013-Finalconditions of climate change, utilization and damage edit-110310.pdf of the ichthyofauna species genetic fund, there is [11] White Paper on the Preparation of the need more attention, a stricter approach and effective Associated Countries of Central and Eastern activities for resolution of their development and Europe for Integration into the Internal Market of conservation problems. the Union, COM(95) 163 final, 3.5.1995 [12] Environmental regulatory reform in the NIS: the 5. References case of the Water sector. Twelfth meeting of the [1] Agenda 21, Rio de Janeiro, 1992. EAP Task Force, 18-19 October 2000, Almaty. [2] Republic of Moldova. Biological Diversity http://www.oecd.org/dataoecd/23/5/2382097.pdf Conservation National Strategy and Action Plan. [13] Guide to the approximation of the European (Ministry of the Environment and Territorial Union Environmental Legislation, SEC (97) 1608 Development. The World Bank), Chişinău, of 25.08.1997. Ştiinţa, 2002, 100 p. http://ec.europa.eu/environment/guide/contents.ht [3] Republic of Moldova, First National Report on m Biological Diversity. (Ministry of the [14] Handbook on the implementation of ec Environment and Territorial Development. The environmental legislation. World Bank), Chişinău, Ştiinţa, 2000, 68 p. http://ec.europa.eu/environment/enlarg/handbook/ [4] Republic of Moldova. Third National Report on handbook.htm the implementation of the Convention on [15] COCIRTA P., CLIPA Carolina. Ecological Biological Diversity. CBD, Chisinau, December, legislation of the Republic of Moldova: Catalogue 2005. of the documents. Chisinau, Stiinta, 2008, 65 pag. [5]http://bsapm.moldnet.md/Text/Raportul%20III/Ra (In Romanian) pr-03-englez.pdf - data of access 4 June 2010 [16] COCIRTA P. Environmental systems and Republic of Moldova. State of the environment electronic information’s in the Republic of Report 2006. Ministry of Ecology and Natural Moldova. Academy of Sciences of Moldova. Resources, Chişinău, 2007, 85 p. Institute of Ecology and Geography – Chisinau, [6] Red Book of the Republic of Moldova, Second 2007. 30 pag. (In Romanian) edition, Stiinta, 2001, 288p. [17]http://www.standardsinfo.net/info/livelink/fetch/2 [7] USATÂI M. “Evolution, conservation, and 000/148478/6301438/index.html sustainable use of diversity of ichthyofauna in 18. http://www.cen.eu/cen/pages/default.aspx aquatic ecosystems of Republic of Moldova”. [19] Catalogue of normative documents in Autoreferat of dissertation for the scientific standardization of the Republic of Moldova. Year degree of doctor Habilitatus in biological 2008. National Institute of Standardization and sciences. Chişinău, 2004, 48 p. (In Romanian) Metrology. Vol.1,2,3. Chisinau, 2008. (In [8] National Strategy of the Sustainable Development Romanian) – “Moldova 21”. Supreme Economic Council [20] http://www.standard.md; under President of the Republic of Moldova, [21] European Red List. - www.iucnredlist/Europe PNUD Moldova, Chişinău, 2000, 129 pag. (In [22]http://ec.europa.eu/environment/nature/legislatio Romanian). n/habitatsdirective/index_en.htm 105 Problems of the harmonizing environmental… / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010) [23]http://europa.eu/legislation_summaries/environm ent/nature_and_biodiversity/l28046_en.htm 106 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 BIODIVERSITY CONSERVATION IN CONSTANŢA COUNTY Silvia TURCU*, Marcela POPOVICI**, Loreley JIANU** *Ovidius University of Constanţa, Doctoral School, Biology Domain, Mamaia Avenue, No. 124, Constanţa, 900552, Romania, sscturcu@yahoo.com ** Environmental Protection Agency Constanţa, Unirii Street, No. 23, Constanţa, 900532 __________________________________________________________________________________________ Abstract: Nature conservation is the action taken by human society to maintain and perpetuation of species of plants and animals. Recognition of the value of biodiversity in Constanta County is done by the special protection of habitats and species for an important number of protected areas. The main instrument governing the activities taking place at the perimeter and adjacent of natural areas is management plan of protected area, in accordance with existing environmental legislation. Keywords: biodiversity conservation, protected areas, administration and custody, Constanţa County. __________________________________________________________________________________________ 1. Introduction Nature Conservation is the action taken by human society to maintain and perpetuation of species of plants and animals [1]. In our country, to provide special measures of protection and biodiversity conservation, was instituted a tiered system of protection, conservation and use, according to the following categories of protected areas: national interest (scientific reserves, national parks, natural monuments, nature reserves, natural parks), the international interest (natural sites of universal natural heritage, geoparks, wetlands of international importance, biosphere reserves), the community interest or Natura 2000 sites (sites of Community Importance, Special Areas of Conservation Areas Special Protection Bird) or local interest [2]. 2. Results and Discussions In Constanţa County, there are over 900 species spermatophytes present, most of these are characteristic species of steppe and forest steppe habitats, over 200 species of vascular flora of national interest, with varying degrees of vulnerability, some of these are endemic species [3]. Fauna of Constanţa County is characterized by great wealth, represented by more than 345 vertebrate taxa - 45 species of mammals, 243 birds, ISSN-1453-1267 19 species of reptiles, 10 species of amphibians and 28 species of fish - and a significant number of invertebrates [3]. In Constanţa County, natural and semi-natural habitats, found in all environments (aquatic, terrestrial and subterranean), are classified into seven classes (coastal and halophilic communities, continental water, scrub and grassland, forests, marshes and wetlands, screes, rock and continental sands and agricultural land and artificial landscapes) which include 58 types of natural habitat and ruderal communities (agricultural land and artificial landscapes) [4]. Thus, since 1970, a number of valuable areas in terms of biodiversity were declared reserves by decisions of Constanţa County People’s Council. In 2000, only two of the previously declared protected natural areas remains areas of local interest (Table 1), for the rest of these, by Law 5/2000 [5] is nationally recognized protected area status. In coming years, new laws were imposed on other areas of protected area status of national interest: Government Decision 2151/2004 [6], Government Decision 1581/2005 [7], Government Decision 1143/2007 [8] and currently totaling 36 protected natural areas of national interest (Table 2). Since joining the European Union in 2007, Romania has emerged as one of the nations that have a true natural heritage, with many protected © 2010 Ovidius University Press Biodiversity Conservation in Constanta County/ Ovidius University Annals - Biology-Ecology Series 14: 107-113 (2010) areas and many species listed in Annexes of Birds and Habitats Directives. Under European Directives, European Council Directive 92/43 EEC [9], and Birds Directive - European Council Directive 79/409 EEC [10], countries of European Union (EU) ensures maintenance or restoration of natural habitats and wild fauna and flora of Community interest in a favorable conservation status, to help maintain biodiversity. Following the transposition of these two Directives into national law was established system of protection for 42 areas: 22 special protection areas for birds (SPA), reported by Government Decision no. 1284/2007 [11] and a number of 20 sites of community importance (SCI), declared by Order no. 1964/2007 [12] (Table 3 and Table 4). There is a part of the “Danube Delta” Biosphere Reserve, internationally protected area, on administrative territory of Constanţa County. This is the largest protected area in the country and has a threefold international status: Biosphere Reserve, Ramsar Site and Site of World Natural and Cultural Heritage (Table 4). “Danube Delta” Biosphere Reserve has its own administrative structure established by Law 82/1993 [13]. Management plan af this protected area was developed by Danube Delta “Biosphere Reserve” Administration. Techirghiol Lake became the Ramsar Site on March 23, 2006 and was classified as wetland of international importance by Government Decision no. 1586/2006 [14] (Table 4). In addition to this status, Techirghiol Lake was declared nature reserve and Bird Protection (Natura 2000). This protected area has not been attributed in custody, but “Dobrogea-Litoral” Water Directorate, in partnership with The Romanian Ornithological Society have developed a management plan for Lake Techirghiol trough project LIFE04NAT/RO/000220 Improving wintering condition for Branta ruficollis at Techirghiol Lake. As can be seen from Tables 1, 2, 3, 4 and 5 responsibilities for managing natural protected areas, placed under special protection and conservation, belong to local authorities for protected natural areas declared by decisions of their, to “Danube Delta” Biosphere Reserve Administration for Biosphere Reserve Danube Delta and to custodians/ administrators for natural protected areas declared by law, by Government decisions or by order of the central public authority for environmental protection. Gaining of custody/administration of natural protected areas is in accordance with the procedure of Government Decision 1533/2007 [15]. However, within six months of the signing of custody agreement for natural protected areas, custodian must develope regulation of protected area, which contains the rules will be respected within the protected area, and within a year to effectuate the protected area management plan, in line with regulation. The measures provided in management plans of protected natural areas are developed taking account of economic requirements, social and cultural as well as on regional and local area, but with priority for the objectives which led to the establishment of protected area. 3. Conclusions Recognition of the value of biodiversity in Constanta county is done by the special care and protection of habitats and species for a number of two protected areas of county interest, 36 protected natural areas of national interest, 42 protected natural areas of interest (Natura 2000 sites): 22 of Special Protection Areas for Birds (SPAs) and 20 Sites of Community Importance (SCI), two natural areas of international concern. Currently, of the 82 protected areas in the county of Constanţa 68 are administered according to law, and 14 will be assumed to custody until the end of 2010. Conservation of biodiversity is in accordance with existing environmental legislation and management plan of protected areas is the main instrument governing the activities taking place at the perimeter and adjacent natural areas. Management of protected natural areas in Constanţa County will improve by developing the management plans, by custodians/ administrators. 4. References [1] BAVARU A. et al., 2007- Biodiversitatea şi ocrotirea naturii, Editura Academiei Române. [2] ***Government Emergency Ordinance no. 57/2007 on the regime of natural protected areas, natural habitats, flora and fauna. 108 Silvia Turcu et al./ Ovidius University Annals, Biology-Ecology Series 14: 107-113 (2010) [3] ***Report on the Environmental Conditions in Constanţa County in 2009. [4] DONIŢĂ N. et al. 2005, "Habitatele din Romania", Editura Tehnică şi Silvică. [5] ***Law 5/2000 approving the national spatial plan and is nationally recognized and protected area status. [6] ***Government Decision 2151/2004 on the establishment of protected area regime to new areas, [7] Government Decision 1581/2005 concerning the establishment of protected area system to new areas. [8] ***Government Decision 1143/2007 concerning the establishment of new protected areas. [9] ***European Council Directive 92/43 EEC on the conservation of natural habitats and wild flora and fauna adopted on May 21, 1992. [10] ***European Council Directive 79/409 EEC on the conservation of wild birds taken on April 2, 1979. [11] ***Government Decision no. 1284/2007 declaring Bird specially protected areas as part of the European ecological network Natura 2000 in Romania. [12] ***Order of Ministry of Environment and Sustainable Development no. 1964/2007 concerning the establishment of protected area system of sites of Community importance, as part of European ecological network Natura 2000 in Romania. [13] ***LAW no 82/1993 establishing Biosphere Reserve “Danube Delta”. [14] ***Government Decision no. 1586/2006 on the classification of protected areas in the category of wetlands of international importance. [15] ***Order no. 1533/2008 approving the Methodology for the award of administration of natural protected areas that require the establishment of administrative structures and methodology for awarding custody of protected natural areas that do not require the creation of management structures. 109 Biodiversity Conservation in Constanta County/ Ovidius University Annals - Biology-Ecology Series 14: 107-113 (2010) Table 1. Natural Protected Areas of county interest No. 1. 2. Protected area “Arborele Corylus colurna” - natural monument “Pâlcul de stejari brumării” - natural monument Administrator Constanţa Hall Mangalia Hall Table 2. Natural Protected Areas of national interest No. 1. 2. 3. 4. 5. 6. 7. Protected area “Acvatoriul litoral-marin Vama Veche-2 Mai” - Zoological and Botanical Reserve “Canaralele din Portul Hârşova” Morfogeological Monument “Cetatea Histria” Scientific Reserve Archaeological Site part of Danube Delta “Biosphere Reserve” “Dealul Alah Bair” - Complex Nature Reserve “Dunele marine de la Agigea” - Botanical Nature Reserve “Grindul Chituc” - Scientific Reserve part of Danube Delta “Biosphere Reserve” “Grindul Lupilor” - Scientific Reserve part of Danube Delta “Biosphere Reserve” 8. “Gura Dobrogei” - Complex Nature Reserve 9. “Lacul Agigea” - Zoological Nature Reserve 10. “Lacul Bugeac” - Complex Nature Reserve 11. “Lacul Dunăreni” - Complex Nature Reserve 12. “Lacul Oltina” - Complex Nature Reserve 13. “Lacul Techirghiol” - Zoological Nature Reserve Administrator/ Custodian National Forest Administration ROMSILVAForestry Department Constanţa Danube Delta “Biosphere Reserve” Administration -Tulcea National Forest Administration ROMSILVAForestry Department Constanţa A.I. Cuza University - Iaşi Danube Delta “Biosphere Reserve” Administration - Tulcea Danube Delta “Biosphere Reserve” Administration - Tulcea National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa - 14. “Lacul Vederoasa” - Complex Nature Reserve “Locul fosilifer Aliman” - Paleontological Monument “Locul fosilifer Cernavodă”- Geological and 16. Paleontological Monument “Locul fosilifer Credinţa” - Paleontological 17. Monument “Locul fosilifer Movila Banului”- Geological 18. and Paleontological Monument 15. 110 National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa Silvia Turcu et al./ Ovidius University Annals, Biology-Ecology Series 14: 107-113 (2010) “Recifii jurasici Cheia” - Geological and Botanical Nature Reserve “Mlaştina Hergheliei” - Complex Nature 20. Reserve “Obanul Mare şi Peştera <La Movile>” 21. Speleological and Morfogeological Nature Reserve National Forest Administration ROMSILVAForestry Department Constanţa The Group of Underwater and Speleological Exploration - Bucharest The Group of Underwater and Speleological Exploration - Bucharest 22. “Pădurea Bratca” - Complex Nature Reserve “Pădurea Dumbrăveni” - Botanical and Zoological Nature Reserve “Pădurea Esechioi” - Botanical and Zoological 27. Nature Reserve “Pădurea Fântâniţa-Murfatlar” - Botanical and 28. Zoological Nature Reserve “Pădurea Hagieni” - Botanical and Zoological 29. Nature Reserve National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa “Pereţii calcaroşi de la Petroşani” - Geological Monument National Forest Administration ROMSILVAForestry Department Constanţa “Peştera <Gura Dobrogei>” - Speleological Monument “Peştera <La Adam>” - Scientific Speleological Reserve “Peştera <Limanu>” - Speleological Monument “Reciful neojurasic de la Topalu” - Geological and Paleontological Monument “Corbu-Nuntaşi-Histria” – Scientific Reserve part of Danube Delta “Biosphere Reserve” “Valu lui Traian Rezervaţie” - Archaeological and Botanical Nature Reserve National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa The Group of Underwater and Speleological Exploration - Bucharest National Forest Administration ROMSILVAForestry Department Constanţa Danube Delta “Biosphere Reserve” Administration -Tulcea 19. “Pădurea Canaraua-Fetii”- Botanical and Zoological Nature Reserve “Pădurea Celea Mare - Valea lui Ene” 24. Complex Nature Reserve 23. 25. “Pădurea Cetate” - Complex Nature Reserve 26. 30. 31. 32. 33. 34. 35. 36. - Table 3. Special Protection Areas – for Birds (SPA) No. Site Name 1. “Aliman – Adamclisi” 2. “Allah Bair – Capidava” Administrator/ Custodian National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa 111 Biodiversity Conservation in Constanta County/ Ovidius University Annals - Biology-Ecology Series 14: 107-113 (2010) 3. 4. “Balta Vederoasa” 5. “Băneasa - Canaraua Fetei” 6. “Canaralele de la Hârşova” 7. “Cheile Dobrogei” 8. “Delta Dunării şi Complexul Razim – Sinoie” 9. “Dumbrăveni” 10. “Dunăre – Ostroave” 11. 12. “Dunărea Veche - Braţul Măcin” 13. “Lacul Dunăreni” 14. National Forest Administration ROMSILVAForestry Department Brăila National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa Danube Delta “Biosphere Reserve” Administration -Tulcea National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa The Group of Underwater and Speleological Exploration - Bucharest EUROLEVEL National Forest Administration ROMSILVAForestry Department Constanţa - “Balta Mică a Brăilei” “Lacul Bugeac” “Lacul Oltina” 15. 16. 17. 18. “Lacul Siutghiol” “Lacurile Taşaul – Corbu” “Lacul Techirghiol” 19. 20. “Marea Neagră” 21. 22. “Stepa Casimcea” “Stepa Saraiu – Horea” “Limanu – Herghelia” “Pădurea Hagieni” Table 4. Sites of Community Importance (SCI) No. 1. Site Name “Balta Mică a Brăilei” 2. 3. “Braţul Măcin” “Canaralele Dunării” 4. “Dealul Alah Bair” 5. “Delta Dunării” 6. “Delta Dunării - zona marină” Administrator/ Custodian National Forest Administration ROMSILVAForestry Department Brăila National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa Danube Delta “Biosphere Reserve” Administration Danube Delta “Biosphere Reserve” Administration 112 Silvia Turcu et al./ Ovidius University Annals, Biology-Ecology Series 14: 107-113 (2010) “Dumbrăveni - Valea Urluia - Lacul Vederoasa” “Dunele marine de la Agigea” “Fântâniţa Murfatlar” 7. 8. 9. National Forest Administration ROMSILVAForestry Department Constanţa A.I. Cuza University Iasi National Forest Administration ROMSILVAForestry Department Constanţa GEOECOMAR 12. “Izvoarele sulfuroase submarine de la Mangalia” “Mlaştina Hergheliei - Obanul Mare şi Peştera Movilei” “Pădurea Esechioi - Lacul Bugeac” 13. “Pădurea Hagieni - Cotul Văii” 14. 15. “Pădurea şi Valea Canaraua Fetii – Iortmac” “Peştera Limanu” 16. 17. 18. “Plaja submersă Eforie Nord - Eforie Sud” “Podişul Nord Dobrogean” “Recifii Jurasici Cheia” 19. 20. “Vama Veche - 2 Mai” “Zona marină de la Capul Tuzla” 10. 11. The Group of Underwater and Speleological Exploration - Bucharest National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa National Forest Administration ROMSILVAForestry Department Constanţa The Group of Underwater and Speleological Exploration - Bucharest EUROLEVEL National Forest Administration ROMSILVAForestry Department Constanţa GEOECOMAR Table 5. Natural Protected Areas of international concern No. 1. 2. Protected Area “Lacul Techirghiol” - Ramsar Site “Delta Dunării” – Biosphere Reserve, Ramsar Site, World Heritage Site Natural and Cultural 113 Administrator Danube Delta “Biosphere Reserve” Administration -Tulcea Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 THE PRESENT SITUATION OF THE NOSE HORNED VIPER POPULATIONS (VIPERA AMMODYTES MONTANDONI BOULENGER 1904) FROM DOBRUDJA (ROMANIA AND BULGARIA) Marian TUDOR Universitatea Ovidius Constanţa, Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole B-dul Mamaia, nr. 124, Constanţa, 900527, România, e-mail __________________________________________________________________________________________ Abstract: Due to the destruction and deterioration of the specific habitats and the increased fragmentation of the remaining ones, the nose horned viper has lost large tracts of vital living space. In addition, road kills, direct kills and collecting by humans contribute to their decline. I tried to estimate the present situation of the nose horned viper populations in Dobrudja, based on literature and our own field data. The main goals were: to investigate the present situation of the nose horned populations in Dobrudja; to identify the most suitable habitats for Vipera ammodytes montandoni; and to locate the viable populations of this viper and current threats to the nose horned viper populations. Keywords: Dobrudja, Nose-Horned Viper, viable populations __________________________________________________________________________________________ 1. Introduction The study of the nose horned viper in general, and of the Dobrudja subspecies in particular, can offer both herpetologists and conservationist biologists important data due to the relatively strict habitat requirements of this herpeto-taxon (particular habitat conditions, the necessary presence of certain prey-species in the habitat etc), as well as to its vulnerability to the modifications of the specific habitats. From this point of view, it is one of the ideal species for monitoring in the protected areas, as well as in those territories to be designated protected areas in the future. The subspecies is considered critically endangered (CR) in the Vertebrates Red List of Romania [1] and it is included in annex 3A of OM 1198/2005 (Species of European interest in need of strict protection, critically endangered species). The populations of Vipera ammodytes montandoni are in a continuous decline [2] due to anthropogenic causes and their need for preservation is all the more imperative as the destruction of the specific habitats has increased considerably over the last few years. 2. Material and Methods ISSN-1453-1267 Starting with 1995, thirty-eight locations mentioned in literature ([3], [4], [5], [6], [7], [8], [9]) in the Romanian area of Dobrudja have been explored with the purpose of verifying the preservation state of the nose horned viper populations. Eight more locations have been explored for the same reason in the Bulgarian region of Dobrudja in 2008. The researches took place especially in spring and autumn, when the vipers are more active and more easily recognizable in the specific habitats [10], [11], [12], [13], [14]. The explorations used visual transects as well as the method of active search in the specific habitats. [11], [15]. The capture and handling of the vipers was accomplished with the help of the herpetological hook and tongs ([16], [17]). Leather gloves were used in the case of small individuals. After identification and sex determination, each individual was released in the same place where it was captured from. Also, the roads that bordered or intersected the explored habitats were repeatedly examined, and all the road kills were photographed and collected. The searches led many times to the discovery of individuals whose death was a result of the direct interaction with © 2010 Ovidius University Press The present situation of the nose horned viper.../ Ovidius University Annals, Biology-Ecology Series 14: 115-120 (2010) humans. In such cases, the vipers had usually been hit to death with stones or other previously mentioned as habitats for nose horned viper populations. hard objects. No instances of natural death were identified among the dead individuals. All the inventoried individuals in each researched habitat were quantified and the determination of the viability degree of the populations was attempted by means of calculating the identified adult/juveniles proportion [18]. The calculation of the viability degree also took into account the state of the habitats and particularly the level of human intervention, starting from the premise that a natural or semi-natural habitat offers much better conditions for the survival of a nose horned viper population than an anthropogenic one. The study has evidenced the fact that in Dobrudja (both the Romanian and the Bulgarian side), the largest populations of Vipera ammodytes montandoni are situated in Dumbraveni Natural Reserve, Babadag Forest, Priopcea Hill, Macin Mountains National Park, Gura Dobrogei Natural Reserve, the ruins of Adamclisi fortress, Canaraua Fetii Natural Reserve, Rusalka, Kaliakra, Bolata Dèrè, Yaillata and Kamen Bryag. Of the total inventoried individuals in the researched areas, 14% were represented by animals whose death was a result of the anthropogenic impact. Among these, 67% are represented by vipers killed deliberately, most having a crushed skull, and 33% are road kills, especially in spring when these reptiles prefer to bask directly on road asphalt (figure 1). 3. Results and Discussions The study has rendered evident certain aspects that complete the data regarding the state of the nose horned viper populations in Dobrudja. Thus, if before our researches, it was considered that Vipera ammodytes montandoni has a relatively large distribution in Dobrudja [7], [19], [9], our data rather bring arguments in favor of the idea that this subspecies currently occupies small habitats in more or less strictly delineated areas. This aspect supports the idea that the exchanges of individuals among populations are very poor or lack completely. This may lead in time to the reduction of the intrapopulation genetic diversity. Also, most of the habitats of nose horned viper populations in Dobrudja are intersected or bordered by roads. Thus, it was observed that, out of a total of thirty-eight locations situated in the Romanian part of Dobrudja where populations of nose horned viper had previously been mentioned [7], only in twenty-five of them (65.8%) the presence of this herpeto-taxon could be rendered evident. Numerous monasteries and hermitages have been built over the past ten years and their presence already generates a rise in the number of direct kills in some locations where there were populations of nose horned viper such as Babadag Forest, Gura Dobrogei, Dumbraveni, Hagieni and the foot of Pricopanului Peak. The existence of this Dobrudja subspecies could no longer be evidenced in the other thirteen locations 33% road kills direct kills 67% Fig. 1. The raport Road kill/Direct kill The estimation of population viability in Dumbraveni Natural Reserve, Babadag Forest, Priopcea Hill, Macin Mountains National Park, Gura Dobrogei Natural Reserve, the ruins of Adamclisi fortress, Canaraua Fetii Natural Reserve, Rusalka, Kaliakra, Bolata Dèrè, Yaillata and Kamen Bryag evidenced the fact that the number of juveniles compared to that of adults is relatively high in these areas, which could thus indicate a high viability of these populations. As a whole, the situation is graphically illustrated in figure 2. In what regards the abundance of individuals in the researched populations, it was observed that in locations such as Gura Dobrogei, Babadag, Dumbraveni, Adamclisi, Hagieni, Canaraua Fetii and Bolata Dèrè, the number of identified individuals is higher (figure 3). Still, this aspect only leads to the 116 Marian Tudor / Ovidius University Annals - Biology-Ecology Series 14: 115-120 (2010) conclusion that these populations might be larger than the ones identified and investigated. Also, this aspect must be correlated with the number of field hours spent in each location. If the number of field The most serious danger for the preservation of this subspecies of horned viper is represented by the destruction of habitats. Immediately after come the road kills and direct kills. The populations of Vipera ammodytes montandoni in Dobrudja are isolated one from the hours spent in the Romanian Dobrudja is approximately equal (generally over 100 hours) in each location, the number of hours spent in the locations of the Bulgarian Dobrudja is much lower (an average of 10-12 hours per location). This is why it is very likely that the number of individuals in the identified populations could be much higher in the Bulgarian locations. Considering the time spent in each location, the relative preservation of the habitats, as well the effort of capturing the animals, all these bring arguments in favor of this hypothesis. Otherwise, in what regards these populations in the Bulgarian side of Dobrudja, the data collected over the 2008 research season evidence a relatively good preservation of the nose horned viper in the natural and semi-natural habitats. No road kills or direct kills were discovered in these areas, probably due to the fact that these habitats are located at a considerable distance from roads and spaces dedicated to activities with anthropogenic impact. Still, given that the data collected here were gathered over a period of only a few months, it is possible that direct kills could occur sporadically due to tourism or animal grazing [20]. At the same time, we estimate that the new buildings, as well as the sale of lands that shelter vipers to investors, will lead to the destruction of their specific habitats in Bulgaria too. In both countries, the expansion of constructions and road improvement with the purpose of easing transport but also of facilitating the access of mass tourism to wild areas, will lead to the enhancement of the anthropogenic impact in areas where it either did not exist or it was sporadic. other, therefore we believe that there are few exchanges of individuals among them or that these exchanges lack completely in some cases, leading thus to the reduction of the intra-population genetic diversity; Our data argument for the existence of at least 12 areas that shelter viable populations of nose horned vipers in Dobrodja. These areas are: Dumbraveni Natural Reserve, Babadag Forest, Priopcea Hill, Macin Mountains National Park, Gura Dobrogei Natural Reserve, the ruins of Adamclisi fortress, Canaraua Fetii Natural Reserve, Rusalka, Kaliakra, Bolata Dèrè, Yaillata and Kamen Bryag. Future studies will focus on the estimation of intra-population genetic diversity and on the dynamics of certain populations of this subspecies in order to propose the best measures intended for the preservation of the Dobrudja nose horned viper. Acknowledgements This study was partly possible thanks to the UNDP/GEF Atlas Project no. 047111 “The strengthening of the national system of protected areas in Romania through the best management practices in the Macin Mountains National Park.” The researches in the Bulgarian area of Dobrudja were possible thanks to the PHARE CBC 2005 Romania-Bulgaria Program RO 2005/017535.01.02.02 “Comparative studies regarding the biodiversity of coastal habitats, the anthropogenic impact and the possibilities for the conservation of important European habitats between Cape Midia (Romania) and Cape Kaliakra (Bulgaria). We are indebted to: Dr. Dan Cogălniceanu and Dr. Marius Skolka for providing references, valuable advice and logistics. Dr. Zsolt Török and Dr. Paul Szekely for support and references. Dr. Olivia Chirobocea for the revising of the text and accurate English translation. 4. Conclusions The main conclusion of the study is that Dobrudja, as biogeographical area well circumscribed and with particular characteristics compared to the other parts of Europe situated at the same latitude, still hosts viable populations of the montandoni horned viper subspecies; 117 The present situation of the nose horned viper.../ Ovidius University Annals, Biology-Ecology Series 14: 115-120 (2010) [12] CAMPBELL, H.W., and S.P. CHRISTMAN 1982 - Field techniques for herpetofaunal community analysis. 193-200 in N. J. Scott, Jr., ed. Herpetological Communities, U.S.D.I. Fish and Wildlife Service, Wildlife Research Report 13, Washington, D.C. 239 . [13] RYAN, T.J., PHILIPPI, T., LEIDEN, Y.A., DORCAS, M.E., WIGLEY, T.B. and 5. References [1] IFTIME, A. (2005) - Reptile. In: Cartea Roșie a vertebratelor României, 173–196. BOTNARIUC ,N. & TATOLE, V. (Eds.). Bucuresti: ed. Curtea Veche.[in Romanian]. [2] GIBBONS, J.W. et. al. 2000 - The Global Decline of Reptiles, Déjà Vu Amphibians BioScience, Vol 50, No.8, 653-666. [3] TOROK, Z.1999 - Note privind distribuția spațiala a herpetofaunei în zona Culmii Pricopanului, Acta oecologica, 6, 57-62. [4] TOROK, Z. 2000 - Șerpii veninoși din România (Venomous snake of Romania), Petarda, no.6, Tulcea, Aves. [5] SOS, T., 2005 - Note preliminare privind distribuția spațiala a herpetofaunei de pe Culmea Pricopanului din Parcul Național Munții Măcin, Migrans, Târgu Mureș, 7(3), 8-10. [6] OTEL, V., 1997 - Investigatii herpetologice în zona munților Măcin și podișul Babadagului, Anal Șt. IDD, 1997, 71-77. [7] FUHN, I.E. & VANCEA, St. 1961 - Reptilia (Țestoase, Șopirle, Șerpi). In: Fauna RPR.Vol. 14(2). Bucuresti: Edit. Academiei RPR. 338 [in Romanian]. [8] MERTENS, R. & WERMUTH, H. (1960) - Die Amphibien und Reptilien Europas. Dritte Liste,nach dem Stand vom 1. Januar 1960. Frankfurt am Main: Verlag Waldemar Kramer. 264. [9] COVACIU-MARCOV S.D, GHIRA I., CICORT-LUCACIU A.D., SAS I., STRUGARIU A., BOGDAN H.V. Contributions to knowledge regarding the geographical distribution of the herpetofauna of Dobrudja, Romania. North-Western Journal of Zoology Vol. 2, No. 2, 2006, 88-125 [10] FUHN, I.E. (1969) - Broaste, serpi, sopirle. Bucuresti: Ed. Natura si Omul. 246 [In Romanian]. [11] COSSWHITE, D.L., S.F. FOX, and THILL R.E. 1999 - Comparison of methods for monitoring reptiles and amphibians in upland forests of the Ouachita mountains, Proceedings of the Oklahoma Academy of Science 79:45-50. [14] GASC, J.-P., CABELA, A., CRNOBRHJAISAILOVIC, J.,DOLMEN, D., GROSSENBACHER, K., HAFFNER, P.,LESCURE, J., MARTENS, H., MARTINEZRICA, J.P., MAURIN, H., OLIVEIRA, M.E., SOFIANIDOU, T.S., VEITH, M. & ZUIDERWIJK, A.(Eds.) 1997 - Atlas of Amphibians and Reptiles in Europe. [15] ENGE, K.M. 2001 - The pitfalls of pitfall traps. Journal of Herpetology 35(3): 467-478. [16] FERNER, J. W. 1979. A review of marking techniques for amphibians and reptiles, Society for the Study of Amphibians and Reptiles, Circular 9: 1-42. [17] KARNS, D.R. 1986 - Field herpetology: methods for the study of amphibians and reptiles, in Minnesota. James Ford Bell Museum of Natural History, occasional papers 18. [18] CORN, P. S., and R. B. BURY. 1990 Reptiles. USDA Forest Service, General and Technical Report PNW-GTR-256, 34. [19] ANDREI, M., 2002 - Contributions to the knowledge of the herpetofauna of southern Dobrudja (Romania). Trav. Mus. Nat. d'Hist. Nat. Gr. Antipa 44, 357-373 [20] BOIAN, P.P. 2007 © Springer, Amphibians and Reptiles of Bulgaria: Fauna, Vertical Distribution and Consrvation, 85-107 , in Biogeography and Ecology of Bulgaria, V. Fet & A. Popov (eds.) 118 The present situation of the nose horned viper.../ Ovidius University Annals, Biology-Ecology Series 14: 115-120 (2010) Rusalka Yailata Kamen Bryag Kaliacra Bolata Dèrè Canaraua Fetii Hagieni Adamclisi Dumbrăveni Babadag Gura Dobrogei Juveniles Măcin Adults Priopcea Niculiţel Târguşor Cerna Albesti Şipotele Casimcea Atmagea Beştepe Cataloi 0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% Fig. 2. The percentage of adults and juveniles in the analyzed populations 119 Marian Tudor / Ovidius University Annals - Biology-Ecology Series 14: 115-120 (2010) Canaraua Fetii Hagieni Adamclisi Dumbrăveni Babadag Gura Dobrogei Măcin Yailata Bolata Déré Kaliakra Priopcea Rusalka Kamen Bryag Niculiţel Târguşor Cerna Albesti Şipotele Casimcea Atmagea Beştepe Cataloi 0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% 14.00% 16.00% Fig. 3. The abundance of Nose-Horned Viper in the analyzed populations 120 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 BODY SIZE VARIATION IN RANA TEMPORARIA POPULATIONS INHABITING EXTREME ENVIRONMENTS Rodica PLĂIAŞU**, Raluca BĂNCILĂ**, Dan COGĂLNICEANU* * Ovidius University Constanţa, Faculty of Natural and Agricultural Sciences, Aleea Universităţii nr. 1, corp B, Constanţa 900470, Romania ** “Emil Racoviţă” Institute of Speleology, 13 Septembrie Road, No. 13, Bucharest 050711, Romania ___________________________________________________________________________ Abstract: We studied the variation in body size in populations of a widespread anuran species, Rana temporaria, from high altitude and latitudes. Our results indicated a variable interannual pattern of body size, suggesting that body size in extreme environments is influenced by many factors. This indicates that long-term series of observations are needed to separate natural fluctuations from man-induced changes. Keywords: Rana temporaria, extreme environments, body size, interannual variation __________________________________________________________________________________________ 1. Introduction During the last decades, many amphibian species have declined from high altitude area, even in habitats apparently without human impact [1, 2]. The causes of some declines remain unknown. Understanding of the life history characteristics of the amphibian populations that inhabit extreme environments at high altitude and latitude is an important step in the evaluation process of the potential causes of decline. Genetic and environmental factors (e.g. temperature, rainfall, trophic resources, competition, predators) determine variation in the life history traits of species occupying a large geographic area [3]. Low temperature, associated with high altitude/latitude, reduces the activity period and the time available for resource exploitation [4]. Temperature affects the duration of hatching and metamorphosis in amphibians. The increase in the adult body size has been frequently associated with a cold annual temperature [5, 6]. Most studies of variation in amphibians body size have focused on latitudinal and altitudinal variation, e.g. trying to establish if the amphibian species follow the Bergmann’s rule [7, 8]. Studies on the interannual variations in amphibians body size generally analyze difference in body condition [9, 10], or variation in age and size at maturity [6]. ISSN-1453-1267 The Common Frog (Rana temporaria) is the most widespread amphibian species in Europe [11]. Its distribution reaches 71o N in Fennoscandia [12] and it can be found even at altitudes of 2600 m [12]. The wide altitudinal and latitudinal range of this species, allows comparisons of life-history traits over a broad range of conditions. In a previous publication we analyzed the altitudinal and latitudinal body size variation among populations from high altitude and latitude of R. temporaria testing if the variation pattern is according to the Bergmann’s rule [13]. In this study we analyzed interannual body size variation in the same Rana temporaria populations, in order to evaluate if the pattern of variation in body size changes in time. We tested the following predictions: i) there is no interannual variation in body size and ii) the mean body size of the frog populations from subarctic regions shows significant variation during a growth season. 2. Material and Methods R. temporaria populations were studied from Kilpisjarvi, Finland (latitude N 69o) in 2003 (August) and 2009 (July), Kolari, Finland (latitude 67.2o) in 2009 (July) and in Retezat National Park, Romania (latitude N 45o) in 2004 (September) and 2009 (August). Latitude and altitude were recorded for each population by using a handheld Garmin GPS. © 2010 Ovidius University Press Body size variation in Rana tempoaria populations / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010) Captured individuals were sexed, weighed (W) to the nearest 0.01 g with a portable electronic balance (AccuLab Pocket Pro), and snout-vent length (SVL) was measured to the nearest 0.5 mm with dialcalipers. Data were log transformed prior to analyses. For comparisons between years and sites we used One-way analysis of variance (ANOVA) and Analysis of covariance (ANCOVA) to compare the slopes of the regression lines. Statistical analyses were performed using SPSS ver. 10.0 (SPSS Inc., 1999). SVL: D = 3.75, p < 0.001). The body size indices of the studied populations are presented in Tables 1 and 2. There was no significant difference in SVL between Retezat National Park and Finland Kilisjarvi populations. We found significant differences in the mean body size indices between the two stations from Finland (Table 3). We then compared W and SVL from different years for the same population. We found significant differences in the interannual variation of the body size indices for juveniles in both Finland and Retezat populations, and in the mean weight for females. Males showed only in Retezat a significant interannual variation in the body size indices (Table 4). We also compared the slopes of the regression lines of W as a function of SVL. The slopes of the regression lines are significantly different for all adults in Retezat and Finland (Fig. 1: F 1,56 = 81.41, p<0.001; Fig. 2: F 1,27 =102.5, p<0.001; Fig. 3: F 1,41 =58.3, p<0.001; Fig.4: F 1,48 =48.4, p<0.001; Fig. 5: F 1,28 =28.8, p<0.001; Fig. 6: F 1,24 =43.1, p<0.001). 3. Results and Discussions A total of 347 individuals were measured and weighed in 2003/2004, of which 237 juveniles and 110 adults (67 males and 43 females) and 157 individuals in 2009 (66 juveniles, 43 females and 48 males). Both log transformed W and SVL were normally distributed (W: D = 5.06, p < 0.001; Table 1. Snout-vent length (SVL) variation according to sex and age classes, in populations of R. temporaria from Finland and Romania in 2009 (FN = Finland - Kilpisjarvi; FS = Finland - Kolari, RNP = Retezat high altitude; SD = standard deviation; Min = minimum; Max = maximum). Females Males Juvenils Populations FS FN RNP Average 71.24 61.15 76.78 SD 6.00 4.15 10.29 Min-Max 62 - 85.6 55.1 - 67.9 55.7-91.8 Average 69.89 62.46 73.41 SD 4.45 3.37 7.41 Min-Max 63.4 - 75 57.8 - 70.6 55.6 - 84.7 Average 42.17 45.95 44.80 SD 11.28 6.95 12.30 Min-Max 28.3 – 59.7 24.2 -55.5 27.8 -59.8 Table 2. Weight (W) variation according to sex and age classes, in populations of R. temporaria from Finland and Romania in 2009 (FN = Finland - Kilpisjarvi; FS = Finland - Kolari, RNP = Retezat high altitude; SD = standard deviation; Min = minimum; Max = maximum). Females Males Juvenils Populations FS FN RNP Average 18.96 14.31 34.69 SD 5.17 4.91 12.49 Min-Max 10.5 - 27.3 9.1 - 24.8 14.07-57.15 Average 17.27 15.85 36.20 122 SD 3.18 4.34 11.40 Min-Max 12.6-22.9 10 - 24.2 13.78 -59.2 Average 4.33 7.78 9.28 SD 3.27 2.85 5.86 Min-Max 1.1 - 10.4 1-13.2 1.7 - 15.04 Rodica Plăiaşu et al. / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010) Table 3. Comparison of SVL and W between the three R. temporaria populations, by using ANCOVA (FN = Finland - Kilpisjarvi; FS = Finland - Kolari, RNP = Retezat high altitude; N = sample size, *P < 0.05, ***P < 0.001, NS = not significant). W FN vs FS Female Male Juveniles FN vs RNP Female N FN 13 17 50 FN 13 Male Juveniles 17 50 SVL 16 9 9 RNP 14 Average FS 18.956 17.267 4.333 FN 14.308 FN 14.308 15.853 7.779 RNP 34.69 22 7 15.853 7.779 36.20 9.28 FS Fa 19.504*** Average FS 71.244 69.89 42.17 FN 61.154 FN 61.154 62.46 45.95 RNP 76.78 17.93*** 5.903* 62.46 45.95 73.41 44.8 6.069* 0.737NS 10.381* Fa 26.384*** 22.88*** 1.748NS 3.627NS 1.778NS 0.198NS Table 4. Comparison of the interannual variation in SVL and W between the three R. temporaria populations, by using ANCOVA (FN = Finland - Kilpisjarvi; FS = Finland - Kolari, RNP = Retezat high altitude; N = sample size, *P < 0.05, ***P < 0.001, NS = not significant). N FN 2003 vs. 2009 Female Male Juveniles RNP 2004 vs. 2009 Female Male Juveniles 2003 29 32 197 2004 14 35 40 2009 13 17 50 2009 14 22 7 W Average 2003 2009 31 14.308 31.76 15.853 3.17 7.779 2004 2009 61.16 34.69 47.14 36.20 2.19 9.28 The variation in the adult body size reported in amphibians can be induced by several factors, including genetic and environmental differences, such as: duration of the activity period, food availability and climatic conditions [6, 14]. Laugen et al. (2005) found that body size decreased with latitude in the Scandinavian Common Frog populations. Comparisons between populations from Western Europe with different activity periods report increases in the mean length, as activity period gets shorter [6]. Rana temporaria populations from the analyzed area show a variable pattern in weight and length. Băncilă et al. (2010) found that latitudinal and altitudinal variation patterns in juvenile body size Fa 4.235* 3.713NS 78.41*** 14.56*** 12.21*** 22.56*** SVL Average 2003 2009 65.94 61.154 67.59 62.46 29.01 45.95 2004 2009 82.7 76.78 77.07 73.41 24.42 44.8 Fa 3.70NS 0.43NS 120.01*** 2.24NS 4.49* 19.4*** were according to the Bergmann’s rule. We found an opposite pattern for juveniles, with decreases in the body size as the activity period gets shorter. Since juveniles have higher growth and development rate than adults, difference could be observed even in the case of short periods of time between sampling. Interpopulational variation in the adult body size could be caused by differences in the age structure. Growth rates in amphibian species can dramatically decrease after the attainment of sexual maturity (e.g. Miaud et al. 1999). Thus, delayed reproduction can allow a prolonged growth period and the attainment of a larger adult size. 123 Body size variation in Rana tempoaria populations / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010) 2.2 2.0 2.0 1.8 W (log) W (log) 1.8 1.6 1.4 1.4 1.2 1.0 1.75 1.6 1.80 1.85 1.90 1.95 RNP 2004 RNP 2009 1.2 RNP 2004 RNP 2009 1.0 1.70 2.00 1.75 1.80 1.85 SVL (log) 1.8 1.8 1.6 1.6 1.4 1.4 1.2 Kilpisjarvi 2003 Kilpisjarvi 2009 1.75 1.80 2.00 2.05 1.2 1.0 1.0 1.70 1.95 Fig. 2. Body size indices for females in RNP populations, 2004 (N=14; R2 = 0.75) and 2009 (N=14; R2 = 0.95). W (log) W (log) Fig. 1. Body size indices for males in RNP populations, 2004 (N=35; R2 = 0.72) and 2009 (N=22; R2 = 0.90). 0.8 1.65 1.90 SVL (log) 1.85 1.90 0.8 1.74 Kilpisjarvi 2003 Kilpisjarvi 2009 1.76 1.78 1.80 1.95 1.82 1.84 1.86 1.88 SVL (log) SVL (log) Fig. 3. Body size indices for females in FinlandKilpisjarvi, 2003 (N=29; R2 = 0.63) and 2009 (N=13; R2 = 0.59). Fig. 4. Body size indices for males in FinlandKilpisjarvi, 2003 (N=32; R2 = 0.70) and 2009 (N=17; R2 = 0.63). 1.6 1.5 1.5 1.4 1.3 W (log) W (log) 1.4 1.3 1.2 1.2 1.1 1.1 0.9 1.70 1.0 Kolari Kilpisjarvi 1.0 1.75 1.80 1.85 1.90 0.9 1.74 1.95 1.76 1.78 1.80 1.82 1.84 1.86 1.88 1.90 SVL (log) SVL (log) Fig. 5. Body size indices for females in Finland Kilspijarvi (N=13; R2 = 0.59) and Finland Kolari 2009 (N=16; R2 = 0.71). Kolari Kilpisjarvi Fig. 6. Body size indices for males in Finland Kilpisjarvi (N=17; R2 = 0.89) and Finland Kolari 2009 (N=9; R2 = 0.70). 124 Rodica Plăiaşu et al. / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010) conservation in Latin America. Conservation Biology, 15: 1213-1223. [3] SORCI G., Clobert J., Belichon S., 1996 Phenotypic plasticity of growth and survival in the common lizard Lacerta vivipara. Journal of Animal Ecology, 65: 781-790. [4] RYSER J., 1996 - Comparative life histories of a low- and a high-elevation population of the common frog Rana temporaria. Amphibia– Reptilia, 17: 183-195. [5] FICETOLA G.F., Scali S., Denoël M., Montinaro G., Vukov T.J., Zuffi M.A.L., PadoaSchioppa E., 2010 - Ecogeographical variation of body size in the newt Triturus carnifex: comparing the hypotheses using an informationtheoretic approach. Global Ecology and Biogeography, 19: 485-495. [6] MIAUD C., Guyétant R., Elmberg J., 1999 Variations in life-history traits in the common frog (Rana temporaria) (Amphibia: Anura): a literature review and new data from the French Alps. Journal of Zoology, 249: 61-73. [7] ADAMS D.C., Church J.O., 2008 - Amphibians do not follow Bergmann’s rule. Evolution, 62: 413-420. [8] ASHTON K.G., 2002 - Do amphibians follow Bergmann’s rule? Canadian Journal of Zoology, 80: 708-716. [9] TOMAŠEVIĆ N., Cvetković D., Aleksić I., Crnobrnja-Isailović J., 2007 - The effect of climatic conditions on post-hibernation body condition and reproductive traits of Bufo bufo females. Archives of Biological Sciences, Belgrade, 59: 51-52. [10] TOMAŠEVIĆ N., Cvetković D., Miaud C., Aleksić I., Crnobrnja-Isailović J., 2008 Interannual variation in life history traits between neighbouring populations of the widespread amphibian Bufo bufo. Revue d’Ecologie (Terre et Vie), 63: 371-381. [11] GASC J.P., Cabela A., Crnobrnja-Isailovic J., Dolmen D., Grossenbacher K., Haffner P., Lescure J., Martens H., Martínez Rica J.P., Maurin H., Oliveira M.E., Sofianidou T.S., Veith M., Zuiderwijk A. (ed), 1997 - Atlas of Amphibians and Reptiles in Europe. Collection Patrimoines Naturels, 29, Societas Europaea Herpetologica, Muséum National d'Histoire Factors such as temperature and humidity can directly affect the activity period and the availability of food, influencing the growth rate and the fat stores; hence they could consequently determine significant interannual variation in the body size. Populations from both analyzed areas exhibit interannual variation in weight and length. This variation mainly affects the weight and could be the result of the differences in the sampling period. The pattern of the adult size variation could also directly result from the variation in the population age structure. Further analyses are necessary to determine whether variation in the age structure are contributing or not to the interannual body size indices. Results suggest that many factors affect the body size in extreme environment and long-term series of observations are needed in order to separate natural fluctuations from the human impact/global warming. 4. Conclusions This study stresses the importance of analyzing interannual variation of life history traits, because one-year data may not properly reflect the features of a population and this issue becomes important in the context of global changes and their possible effects on the amphibian populations. Acknowledgements The research was funded by the EU FP6 (Lapbiat) and EU FP7 (Lapbiat 2) Romanian CNCSIS grant 1114/2004. We are grateful to Claudia Jianu, Dorel Ruşti, Ioan Ghira and Marian Tudor for their help during fieldwork. 5. References [1] LAURANCE W.F., McDonald K.R., Speare R., 1996 - Epidemic disease and the catastrophic decline of Australian rain forest frogs. Conservation Biology, 10: 406-413. [2] YOUNG B.E., Lips K.R., Reaser J.K., Ibanez R., Salas A.W., Cedeno J.R., Coloma L.A., Ron S., La Marca E., Meyer J.R., Munoz A., Bolanos F., Chaves G., Romo D. 2001. Population declines and priorities for amphibian 125 Body size variation in Rana tempoaria populations / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010) populations. Studii şi Cercetări, Biologie, Universitatea din Bacău,17: 43-46. [14] MORRISON C., Hero J., 2002 - Geographic variation in life history characteristics of amphibians: a review. Journal of Animal Ecology, 72: 270-279. [15] LAUGEN A.T., Laurila A., Jönsson K.I., Söderman F., Merilä J., 2005 - Do common frogs (Rana temporaria) follow Bergmann’s rule? Evolutionary Ecology Research, 7: 717-731. Naturelle & Service du Petrimone Naturel, Paris, 496 pp. [12] MERILÄ J., Laurila A., Laugen A.T., Rasanen K., Pahkla M., 2000 - Plasticity in age and size at metamorphosis in Rana temporaria comparison of high and low latitude populations. Ecography, 23: 457-465. [13] BĂNCILĂ R.I., Plăiaşu R., Cogălniceanu, D., 2010 - Effect of latitude and altitude on body size in the common frog (Rana temporaria) 126 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 UTILIZATION OF EPIFLUORESCENCE MICROSCOPY AND DIGITAL IMAGE ANALYSIS TO STUDY SOME MORPHOLOGICAL AND FUNCTIONAL ASPECTS OF PROKARYOTES Simona GHIŢĂ**, Iris SARCHIZIAN*, Ioan ARDELEAN*** Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology, Mamaia Avenue, No. 124, Constanţa, 900552, Romania, e-mail: ghitasimona@aim.com, irissarchizian@yahoo.com ** Constanta Maritime University, Department of Environmental Engineering, Mircea cel Batrin, No. 104, Constanta, 900663, Romania, e-mail:ghitasimona@aim.com; *** Institute of Biology, Splaiul Independenţei, No. 296, Bucharest, 060031, Romania, email:ioan.ardelean57@yahoo.com * __________________________________________________________________________________________ Abstract: The aims of this study is to argue, based on original results, the importance of utilization of epifluorescence microscopy to study some morphological and functional aspects of prokaryotes allowing to perform total cell counts , direct viable count count, count of permeabilised cells, chlorophyll containing cells or putatively capsulated cells. Automated image analysis of the results thus obtained was done using CellC and ImageJ software which allow the quantification of bacterial cells from digital microscope images, automated enumeration of bacterial cells, comparison of total count and specific count images, providing also quantitative estimates of cell morphology. Keywords: epifluorescence, digital image analysis, heterotrophic bacteria, cyanobacteria. __________________________________________________________________________________________ 1. Introduction The use of epifluorescence microscopy to study different aspects of prokaryotes at population and single cell level significantly improved the knowledge concerning which species are present in a given sample, the cell density and the metabolic statues of the population as a whole or of each single prokaryote cell (Van Wambeke, 1995; Manini & Danovaro, 2006; Falcioni et al., 2008; Kirchman, 2008; Ardelean et al., 2009). In the last decades there is also an increase in the development and use of different softwares for automated analysis of the digital images thus obtained (Ishii et al. 1987; Estep & Macintyre 1989; Embleton et al., 2003; Walsby, 1996; Congestri et al. 2003; Selinummi et al., 2005, 2008). The aims of this study is to argue, based on original results, the importance of utilization of epifluorescence microscopy coupled with automated image analysis to study some morphological and ISSN-1453-1267 functional aspects of prokaryotes allowing to perform total cell counts (acridine orange, DAPI, SYBR Green 1), direct viable count (elongated cell in the presence of nalidixic acid, labelled with acridine orange), count of permeabilised cells (cells permeable to propidium iodide), putatively capsulated cell (labelled with aniline blue) and chlorophyll containing cells both in enriched cultures and in natural (microcosms) samples. 2. Material and Methods A. Study area and sampling. Samples were collected in sterile bottles in October 2008 and May 2009 from sulphurous mesothermal spring (Obanul Mare) placed in north of Mangalia City (43˚49’53.6’’N; 28˚34’05.3’’E). The samples were divided in sub-samples, one being immediately fixed with buffered formaldehyde (2% final concentration) and the second one used to isolate cyanobacteria by inoculation into conical flasks with either BG 11 © 2010 Ovidius University Press Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) medium or nitrate - free BG 11 medium (BG 0 ) (Rippka et al., 1979). Another series of natural samples were collected from Black Sea (Tomis seaport at 0.5m depth; 44o10’44’’N; 28o39’32’’E) in March 2009. B. Culture conditions. Natural samples inoculated in either BG 11 or BG 0 media, either solid of liquid, were incubated in culture room at 25 ± 1ºC and illuminated with fluorescent tubes having the photon rate of 50 μmol m–2 s–1 at surface of the culture vessels. C. Microcosms. Taking into account the advantages of microcosms (Iturbe et al., 2003; Molina-Barahona et al., 2004) we used this opportunity as previously (Ardelean et al., 2009). D. Total cell count (AO; DAPI, SYBR Green I) Total bacterial count were performed using acridine orange, DAPI and SYBR Green I (Luna et al., 2002; Lunau et al., 2005; Manini & Danovaro, 2006). For AO and DAPI (5 μg/mL dye final concentrations) subsamples were stained for 5 minutes and were filtred on black Millipore 0,22µm pore size filters. Unlike AO, using DAPI for bacterial visualization and enumeration has the advantages of low background fluorescence and that DAPI stains only DNA. For SG (1µL/10µL sample final concentrations) subsamples were stained for 10 minutes and were filtred on black Millipore 0,22 µm pore size filters. Color filters were washed with 10 ml of 17 ‰ saline solution. SG as a permeant DNA-binding stain and determine the total fraction of cells from natural samples. E. Permeabilized (dead) cells (PI+) PI is a double-charged phenanthridium derivative and is one of the most common stains for dead cells (Luna et al., 2002). PI is thus assumed to be unable to penetrate cell membranes. In our natural samples we used a PI concentration of 5 μL/ml sample. Also stained samples were filtered through black Millipore 0,22 µm pore size filters and then inspected under a epifluorescence microscope. The disruption of planktonic cell aggregates for cell enumeration were done as previously shown (Ardelean et al., 2009). F. Enumeration of (putatively) capsulated cells (AB+). Cell capsule was also inspected using aniline blue (AB) which is a fluorescent dye specific which seems to be specific for 1,3 beta glucans (Hong et al., 2001) found in plants and as capsular material in many microorganisms (Nakanishi et al., 1976; McIntosh et al., 2005). Capsular envelopes are widely distributed in marine free-living and particleassociated bacteria (Heissenberger et al., 1996) and are a signature of active bacteria (Stoderegger & Herndl, 2002). Bacteria with an intact intracellular structure, and therefore potentially active bacteria, are surrounded by a capsular layer, while the vast majority of bacteria with a damaged structure lack such a capsule (Heissenberger et al., 1996). Laboratory experiments indicated that active bacteria are constantly renewing their capsular envelope and releasing a significant fraction of the polysaccharide layer into the ambient water (Stoderegger & Herndl, 2002). The samples were treated with AB (5 µg/mL final concentration) for 5 minutes and then filtered and counted as shown above for AO staining . G. The automatic cell analysis were done with two software ImageJ and CellC, who was applied to digital images of whole cells color-stained bacteria and cyanobacteria. The analysis proceeds few important steps: the background is separated from the objects based on the intra-class variance threshold method; noise and specks of staining color in the image can affect the reliability of the analysis, so those was removed. The removal was done applying mathematical morphology operations to the image; then separation of clustered objects was performed (Selinummi, 2008). The length of cells was determined with ImageJ software using a calibration scale. H. Cyanobacteria (natural fluorescence) Visualization of hydrocarbon tolerant phototrophic microorganisms, also for unicellular or filamentous cyanobacteria from sulphurous mesothermal spring; chlorophyll a in natural environments (either marine or spring) was done using an epifluorescence microscope (N-400FL, lamp Hg 100W, type on the blue filter; Sherr et al., 2001) as previously shown (Ardelean et al., 2009). I. Direct viable count (cells capable of division) is based on the Kogure method developped 128 Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) by incubation of samples with a single antimicrobial agent (nalidixic acid) and nutrients (yeast extract). Nalidixic acid acts as a specific inhibitor of DNA synthesis and prevents cell division without affecting other cellular metabolic activities, including cell growth; thus viable cells growth but do not divide, thus becoming longer/larger than cells unable to grow (Kogure et al., 1979). Experiments were done with 40mL samples from each microcosms in which the sample was filtered through 0.45 µm filter (2 and 3) supplemented with yeast extract (50 mg / L final concentration), nalidixic acid (20 mg / L final concentration) (Kogure et al., 1979) and gasoline (0.5% final concentration); 17 hours before the start of experiment all samples were kept in an incubator at a temperature of 30oC and continuous stirring. Subsequently samples were incubated under the conditions previously reported and samples were harvested each two hours (considering the time T o , T 1 –after 2 hours, T 2 - after 4 hours; T 3 - after 6 hours, T 4 - after 8 hours). 3. As shown in figure 1 the total number of heterotrophic cells counted using AO or DAPI is practically the same. Quantification was performed on samples previously fixed in experimental microcosms (M1 and M2). Comparing the total number of bacterial cells obtained with AO and DAPI stained (20 μL/mL sample) in experimental microcosms, we have shown that there are no significant differences in the use of two fluorochromes on natural samples (M1: 13.719,5 cells ml-1 – SD (±39,7) AO and 13.494,6 cells ml-1 SD (±22,2) DAPI, respectively M2: 14.619,1 cells ml-1 – SD (±28,8) AO and 15.787,4 cells ml-1 SD (±32,8) DAPI). Results and Discussions 1. Total count cell (AO+, DAPI+), permeable (dead) cells (PI+) and (putative) capsulated cells (AB+) In experimental microcosms we viewed the gasoline tolerant/oxidizing bacteria to make a clear distinction between the total number of cells (stained with AO, DAPI), the number of encapsulated, active cells, (AB +), and the number of permeable (PI+) , dead (figures 1 and 3). Fig 2. Total number of cells obtained using AO and SG in natural sample To assess the number of cells obtained after staining AO and SG, we used unfixed samples collected from microcosm 1. As shown in figure 2 there are differences in total counts obtained by the use of either AO or SYBR Green 1, the higher count obtained with the last fluorochrome (47,6%) being due to its higher fluorescence yield, in agreement with international literature (Weinbauer et al., 1998; Luna et al., 2002; Lunau et al., 2005 ), allowing the visualization of smaller cells. As shown in figure 1, the number of dead cells (PI positive) is 12.3% of the total number obtained with the two fluorochromes, AO and DAPI. In figure 3 one can see the cell densities of putative capsulated cells which are 10% from the density obtained with acridine orange. Fig 1. Comparison between the total cell count (AO+ and DAPI+) and permeable cells density (IP+) 129 Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) Fig 5. Filaments of cyanobacteria in the M2. 3. Direct viable count In figure 6 there are presented the results concerning changes in average cell lengths of bacterial populations from the two microcosms with filtered water (0,45µm) each supplemented with yest extract, nalidixic acid and gasoline (see Materials and methods). Fig 3. Aniline blue positive cells as compared with acridine orange positive cells. 2. Cyanobacteria (natural fluorescence) Natural fluorescence of these prokaryotic in various natural environments (marine and sulphurous mesothermal spring) and marine microcosms was studied by epifluorescence microscopy (figure 4). a b c d Fig 6. Average length of cells from T o to T 4 (after 8 hours of incubation with nalidixic acid) in the two microcosms (M2 and control, M3) Fig 4. Natural fluorescence of gasoline-tolerant oxygenic phototrophic microorganism from microcosm 2 supplemented with gasoline (a); microcosm 1 supplemented with gasoline and nutrient (b) and microorganism isolated from sulphurous mesothermal spring Obanul Mare (Mangalia) (c and d). As can be seen in Figure 6, after 8 hours of incubations, the average length of M2 cells is about 7 µm, compared with the M3 where the cells were maintained in high proportion in the form of cocci (average diameter of about 2 µm). These results argue the possibility to count viable cells, cells able to grow, by a relatively simple method. It seems appropriate to assume that the large increase in cell size in bacterial populations which have been previously selected to grow in the presence of gasoline (microcosms 2) is due to the cells ability to oxidize/tolerate gasoline, as compared with the populations sampled from the control microcosms where the proportion of gasoline tolerant bacteria is In microcosm 1 cyanobacteria filaments are much thinner (1.35 ± 0.27) compared with microcosm 2 (3.87 ± 0.57) (fig.5); the significance of this difference being under investigation. 130 Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) very low (and responsible for the low increase in the average cell lengths in M3). In M3 (control) one can see a rather constant length of some cells during incubation (2,15±0,37) whereas in M2 there was a sudden increase in cell length (6,46±1,54) to the time T 2 (4 hours incubation) then there was a steady increase until T 4 (8 hours incubation). In Figure 7 are some random fields of cells in the two microcosms to highlight how cell elongation occurred from the T o to T 4 only in M2. a b c d For study bacteria and cyanobacteria from our samples ImageJ was the main software for measure the length of cells and pixel value statistics of userdefined selections, creating density histograms and line profile plots, supports standard image processing functions such as contrast manipulation, sharpening, smoothing, edge detection and median filtering. Digital images are two-dimensional grids of pixel intensities values with the width and height of the image being defined by the number of pixels in x (rows) and y (columns) direction. Thus, pixels (picture elements) are the smallest single components of images, holding numeric values – pixel intensities – that range between black and white (ImageJ user guide). Microphotographs used in this study was RGB images, RGB/HSB stacks, and composite images. People can see color with significant variations and the popular phrase “One picture is worth ten thousand words” may not apply to certain color images, especially those that do not follow the basic principles of Color Universal Design. That why this combining digital image analysis and automated analysis methods was usefull to distinguish some morphological and functional aspects of prokaryotes. We displied with ImageJ simultaneously several selections or regions of interest named ROIs, who can be measured, drawn or filled. Selections was initially outlined in one of the nine ImageJ default colors (Red, Green, Blue, Magenta, Cyan, Yellow, Orange, Black and White) and then, once created, selections was contoured or painted with any other color. Most of ImageJ analyses was printed to the Results table. Fig 7. Evaluating cell elongation in microcosm 2 (aT o and b-T 4 ) respectively in the control microcosm (c- T o and d-T 4 ) Digital Image Analysis and automated image analysis for epifluorescence The automated approach will not only remove the need for tedious manual analysis work, but also enable biologists to measure cellular features not feasible by the standard manual techniques (Selinummi, 2008). In our studies we used ImageJ software - a public domain Java image processing and analysis program inspired by NIH Image for the Macintosh, who runs, either as an online applet or as a downloadable application, on any computer with a Java 1.5 or later virtual machine. This software was used to display, edit, analyze, process, save and print 8–bit, 16–bit and 32–bit epifluorescence digital images, many image formats including TIFF, GIF, JPEG, BMP, supporting ‘stacks’and hyperstacks, a series of images that share a single window. Fig 8. The ImageJ Window (http://rsbweb.nih.gov/ij/). Straight Line Selection with “Alt” from computer keeps the line length fixed while moving either end of the line and forces the two points that define the line to have integer coordinate values when creating a line on a zoomed image. The CellC software is the second software used in automated analysis of our microscopy images like cell enumeration and measurements of cell’s properties (size, shape, intensity). We applied the 131 Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) algorithms of CellC software for digital images, because this have three important parts: a MATLAB figure file of the segmented image (this can be exported in any common image file format; a comma separated value (CSV) - file with quantitative data of the cells (was opened in a spreadsheet program Excel for further analysis); a summary CSV-file with the cell count for each of the analyzed images for a quick overview of the analysis process (this file were only saved in the batch processing mode). Fluorescence microscopy digital images were analyzed and the objects has different intensity than the background. Commonly, this property holds true for images of bacteria (http://sites.google.com/site/cellcsoftware/). included graphical user interface, and the batch processing mode enables fast and convenient processing of hundreds of cell images. CellC enumerate bright cells on a dark background (epifluorescence). We also used two different methods to process the images: one image/image pair at a time; several images pairs sequentially in batch processing mode. If the background of the image is uneven (because of e.g. misaligned lighting), it is preferable to choose this option. The default option in CellC is to present the measured parameters in pixels. By checking this box we define how many micrometers one pixel corresponds to, and receive all measurement results in micrometers. The correct value of this setting obviously depends on the imaging setup, such as on the camera and the objective, and must be determined outside CellC, using ImageJ to calibrate the scale. The main technical requirement for using CellC is the clear visual distinction between the cells to be counted and their background, which could be achieved relatively easy by epifluorescence microscopy (Ardelean et al., 2009). If darker regions exist inside cells, thresholding may result in false holes inside cells (darker pixels are considered background). By selecting this option, these holes are automatically filled. Sometimes the fill can cause worse cell cluster separation results. Automatic removal of over/undersized cells were selected, because CellC automatically decides which particles are too small to be considered as real cells. All detected objects that are smaller than 1/10 of the mean size of all objects, were removed. Because the sizes of under/oversized particles were known using “Analyze Measure” option of ImageJ, it was possible to set the thresholds manually by using the text boxes. The unit of sizes depends on the user defined unit (pixels/μm2). The CSV data sheet consists of following columns: cell's serial number (a unique number given to each cell); area of cell (estimate of the cell area); approximate volume (approximation of the volume of the cell); length (estimate of the cell length); width (estimate of the cell width); intensity mean, (mean intensity of the cell); intensity maximum, (maximum intensity of the cell); solidity (estimate of the shape of the cell); compactness (estimate of the shape of the Fig 9. CellC’s interface (http://sites.google.com/site/cellcsoftware/) used for automated digital analysis of bacteria/cyanobacteria. Furthermore, CellC software were used for two important purposes: to calculate total object count (e.g. DAPI stained cells) and co-localization analysis, comparing total and specific count images of the same location. When two images were analyzed, the co-localization was measured by comparing which cells are present only in the first image, and which are visible in both of the images. The binarized result images was saved as JPG-images, and the enumeration results and statistics are saved as an Excel-ready CSV file. The images was processed one at a time, or automatically in a batch. Graphical illustration of the analysis process and a part of a CSV-file opened in a spreadsheet program are given in Figure 9. The CSV-file gives, for each cell in the image, size and intensity information as well as information on cell morphologies. All results produced with digital image processing algorithms are perfectly reproducible. The image processing methods used guarantee that all images are analyzed using the same criteria, and therefore results between different images are comparable. CellC software is easy to use due to the 132 Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) cell). Means of each column and the unit of measure (pixels or micrometers) are presented in the end of the file. Image acquisition—Images for analysis were done with a Canon digital camera. Brightness and contrast were adjusted for the first image and kept unchanged throughout the image acquisition procedure. The images (1600 by 1200 pixels, 256 dpi) were acquired at 50x magnification and stored as 543-KB JPG files. Additional images acquired at 100x magnification were used to verify that measurements of individual filaments/ bacteria were independent of magnification a) Acridin-orange stained filamentous cyanobacteria isolated from mesothermal sulfurous spring were analysed using Image J software for distinguish heterocystous cells. First of all, the original RGB image (Figure 10 A) were transformed into 32-bit images, then we adjust the brightness/contrast and also applied smooth or find edges (Figure 10 B) option from processing images. The same image were analysed with CellC software (Fig. 10 C) to count the cells from filamentous cyanobacteria or to measure the size of each cells. calibrated eyepiece graticule as reference (Ardelean et al, 2009). Digital images from AO staining filaments of cyanobacteria in microcosm were treated with ImageJ to distinguish the heterocystous cell. Fig 11. Cyanobacteria with heterocyst presence in microcosm 2; AO staining (arrow indicate heterocyst cell present in samples of microcosm supplemented with gasoline). To avoid uncertain estimates of filament length and width, the number of filaments presented in one image should not be too high. Extreme filament densities would undoubtedly increase filament overlap and lead to uncertain measurements unless samples are diluted (Almesjö & Rolff, 2007). We use a blue light epifluorescence filter set to visualize AO-stained bacteria (N-400FL type). AO stains both DNA and RNA so is used for the enumeration of total bacteria. In figure 12 A we present only an example of digital analysis of fig.7 b: first, we adjust contrast/brightness of digital image, then analyse measure of graticula presented in fig. 7b and set the calibration bar to determine correctly the length of each bacteria treated with nalidixic acid. In B is presented image analysis using CellC software. B A C Fig 10. A – digital image of heterocystous cyanobacteria isolated from sulphurous mesothermal spring Obanul Mare (Mangalia) stained with AO; B – find edges of panel A using ImageJ software; C- total count analysis of panel A using CellC software (48 cells counted from cyanobacteria’s filaments). B A Fig 12. Image analysis program Image J (A) and CellC (B) from microcosm 2, elongated cells at time T 4 (8hours) Validation of any count were done using manual count. ImageJ software were used for automated measuring cell’s length (µm), using a b) DAPI were used to view filamentous cyanobacteria isolated from mesothermal sulfurous 133 Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) c) Aniline blue is highly specific for staining type polysaccharide. Use of aniline blue is a good method not only for detection of production of exocellular β-1,3-glucan, but also for detection of some β-glucan in the cell wall (Nakanishi et al., 1976). In figure 15 is apparent the AB stained heterotrophic cells from microcosm supplemented with gasoline and cyanobacteria cells from microcosms and sulphurous spring samples. spring and heterotrophic and phototrophic bacteria from marine environment. The fluorochrome DAPI is the most commonly bacterial stain for a wide range of sample types. DAPI is a nonintercalating, DNA-specific stain which fluoresces blue or bluish-white (at or above 390 nm) when bound to DNA and excited with light at a wavelength of 365 nm (Kepner & Pratt, 1994). When unbound, or bound to non-DNA material, it may fluoresce over a range of yellow colors (see figure13). DAPI-stained filaments of cyanobacteria isolated from sulphurous spring Obanul Mare (Mangalia) reveal heterogeneous cells as can be seen in Fig.13a. a b c Fig 15 . Visualisation of encapsulated bacteria and cyanobacteria after aniline-blue staining on M2 (a), M1 (b) and from sulphurous spring samples (c). d) PI staining is generally used for the evaluation of plasma membrane integrity by fluorescence. Literature mentions that molecular weighs PI is 668,4 and is thus assumed to be unable to penetrate cell membrane (Manini & Danovaro, 2006). In figure16 living bacteria appeared green due to the excitation of the AO dye with which the cells have been stained and the samples stained with PI and appear red fluorescent cells; the bacteria were counted under blue excitation. a b Fig 13. DAPI-stained cyanobacteria isolated from sulphurous spring (a), arrows indicates septa between cells ; bacteria/cyanobacteria isolated from marine environment (b); both a and b treated with Image J and CellC software. a b c Fig 16. Marine bacteria examined using epifluorescence microscopy (magnification x1000), Fig (a) illustrate bacteria stained with propidium iodide (dead cells) in microcosms 1 and (b) respectively microcosms 2; total count analysis using the CellC software (c). Fig 14 . Cyanobacteria and heterotrophic cells in microcosm supplemented with gasoline/M2 - DAPI stain 134 Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) Throughout the investigations conducted continuously attempted to determine the nature of connections between communities of microorganisms and how and to which condition each. e) Natural fluorescence - In figure 17 we describe succesfully separatation with ImageJ of cells by natural fluorescence of photosynthetic gasolinetolerant/oxidant microorganisms isolated from mesothermal sulphurous spring in different chanell – red and green- and then each image were automat counted with CellC, obtaining finally the number of cells red and green separately. 4. The utilization of epifluorescence microscopy and digital image analysis enable us to study some morphological and functional aspects of prokaryotes: total cell counts (acridine orange, DAPI, SYBR green 1), direct viable count (elongated cell in the presence of nalidixic acid, labelled with acridine orange), count of permeabilised cells (cells permeable to propidium iodide), capsulated cell (labelled with aniline blue) and chlorophyll containing cells, both in enriched cultures and in natural / microcosms samples. The total number of heterotrophic cells counted using AO or DAPI is practically the same whereas total counts obtained with SYBR Green 1 are 47,6% higher. The number of dead cells (PI positive) and that of (putative) capsulated cells are 12.3% and 10%, respectively of the total number ( AO and DAPI). The image analysis systems presented here was performed for counting and estimating the length of bacteria/cyanobacteria with uniform morphology. The presented methods does not totally exclude the need for manual microscope analyses of water samples, and automated procedures must intermittently be validated by independent manual procedures. Fig 17. Natural fluorescence of (A) photosynthetic gasoline-tolerant/oxidant microorganisms isolated from mesothermal sulphurous spring and digital image analysis of chlorophyll autofluorescence in (B) green channel ; (C) red channel and (D-E) total count analysis of red/green channels using the CellC software. In figure 18 there are presented images showing the natural fluorescence of chlorophyll, as an image of marine oxygenic gasoline tolerant/ oxidant phototrophic microorganisms. Difference is clearly apparent width of filaments of cyanobacteria developed in the experimental microcosms. The measurements were performed with Image J program as shown previous (see point 2). a Conclusions Acknowledgment We are grateful to Dr. Tech. Jyrki Selinummi (Department of Signal Processing, Tampere University of Technology, Finland) for very useful and kind advices concerning the use of software CellC and ImageJ. b Fig 18. Autofluorescence of chlorophyll from oxygenic photosynthetic microorganisms: microcosms 1 (a) and 2 (b). 5. Epifluorescence techniques and image analysis has increasingly been used to determine cell size, bacterial abundance and detection of physiological characteristics like damaged versus intact cell membranes. References [1] VAN WAMBEKE F., 1995- Numeration et taille des bacteries planctoniques au moyen de l`analise d`images couplee a l`epifluorescence. Oceanis, 21: 113-124. 135 Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) [2] MANINI E. & DANOVARO R., 2006- Synoptic determination of living/dead and active/dormant bacterial fraction in marine sediments. FEMS Microbiol. Ecol, 55: 416-423. [3] FALCIONI T., PAPA S., GASOL J.M., 2008Evaluating the flow-cytometric nucleic acid double-staining protocol in realistic situations of planktonic bacterial death. Appl. Environ. Microbiol, 74: 1767-1779. [4] KIRCHMAN D.L., 2008- Microbial Ecology of the Oceans. John Wiley & Sons, NY, 593. [5] ARDELEAN I.I., GHIŢĂ S., SARCHIZIAN I., 2009-Epifluorescent method for quantification of planktonic marine prokaryotes, Proceedings of the 2nd International Symposium “New Research in Biotechnology” serie F: 288-296. [6] ISHII T., ADACHI R., OMORI M., SHIMIZU U., IRIE H., 1987 - The identification, counting, and measurement of phytoplankton by an image-processing system. J. Cons. Ciem., 43:253-260. [7] ESTEP K. W. & MACINTYRE F., 1989 Counting, sizing, and identification of algae using image analysis. Sarsia, 74: 261-268. [8] EMBLETON K. V., GIBSON C. E., HEANEY S. I., 2003 - Automated counting of phytoplankton by pattern recognition: a comparison with a manual counting method. J. Plankt. Res., 25: 669-681. [9] WALSBY A. E. & AVERY A., 1996 Measurement of filamentous cyanobacteria by image analysis. J. Microbiol. Meth., 26:11-20. [10] CONGESTRI R., CAPUCCI E., ALBERTANO P., 2003 – Morphometric variability of the genus Nodularia (Cyanobacteria) in Baltic natural communities. Aquat. Microb. Ecol. 32: 251-259. [11] SELINUMMI J., SEPPÄLÄ J., YLI-HARJA O., PUHAKKA J.A., 2005 - Software for quantification of labeled bacteria from digital microscope images by automated image analysis”. BioTechniques, 39: 859–863. [12] SELINUMMI J., 2008- On Algorithms for Two and Three Dimensional High Throughput Light Microscopy, Ph.D Thesis for the degree of Doctor of Technology. [13] RIPPKA R., DERUELLES J., WATERBURY J.B,. HERDMAN M., STANIER R.Y., 1979 - [14] [15] [16] [17] [18] [19] [20] [21] 136 Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of Geneneral Microbiology., 111: 1 61. ITURBE R., FLORES R.M., TORRES L.G., 2003- Subsoil polluted by hydrocarbons in an out-of-service oil distribution and storage station in Zacatecas, Mexico. Environ Geol, 44: 608–620. MOLINA-BARAHONA L., RODRI´GUEZVA´ZQUEZ R., HERNA´NDEZ- VELA´SCO, C. VEGA-JARQUIN, O. ZAPATA-PE´REZ M., MENDOZA- CANTU´ A., ALBORES A., 2004- Diesel removal from contaminated soils by biostimulation and supplementation with crop residues, Appl Soil Ecol, 27: 165–175. LUNA G.M., MANINI E., DANOVARO R., 2002- High fraction of dead and inactive bacteria in coastal marine sediments: comparison of protocols and ecological significance. Appl. Environ. Microbiol, 68: 3509-3513. LUNAU M., LEMKE A., WALTHER K., MARTENS-HABBENA W., SIMON M., 2005An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy. Appl. Environ. Microbiol, 7: 961-968. HONG Z., DELAUNEY A.J., VERMA D.P.S., 2001-A cell plate-specific callose synthase and its interaction with phragmoplastin.Plant Cell, 13:755-768 NAKANISHI I., KAZUTSUGU K., SUZUKI T., ISHIKAWA M., BANNO I., SAKANE T., HARADA T.,1976- Demonstration of curdlantype polysaccharide and some other β-1, 3glucan in microorganisms with aniline blue. J. Gen. Appl. Microbiol., 22: 1-11. MC INTOSH M., STONE B. A.,. STANISICH V. A,, 2005-Curdlan and other bacterial (1-3)-βD-glucans. Appl Microbiol Biotechnol, 68: 163173. HEISSENBERGER A., LEPPARD G.G., HERNDL G.J., 1996 - Relationship between the intracellular integrity and the morphology of the capsular envelope in attached and free-living marine bacteria. Appl. Environ. Microbiol., 62: 4521-4528. Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) [22] STODEREGGER K.E. & HERNDL G.J., 2002 Distribution of capsulated bacterioplankton in the North Atlantic and North Sea. Microb. Ecol., 44: 154-163. [23] SHERR B., SHERR E.B., DEL GIORGIO P., 2001- Enumeration of total and highly active bacteria. Methods in Microbiology, 30: 129161. [24] KOGURE K., SIMIDU U., TAGA N., 1979- A tentative direct microscopic method for counting living marine bacteria. Can.J.Microbiol, 25:415-420. [25] WEINBAUER M.G., BECKMANN C., HOFLE M.G., 1998 – Utility of green fluorescent nucleic acid dyes and aluminium oxide membrane filters for rapid epifluorescence enumeration of soil and sediment bacteria. Appl. Environ Microbiol, 64: 5000-5003. [26] http://rsbweb.nih.gov/ij/ [27] http://sites.google.com/site/cellcsoftware/ [28] ALMESJÖ L. & ROLFF C., 2007 - Automated measurements of filamentous cyanobacteria by digital image analysis. Limnol.Oceanogr. Methods, 5: 217-224. [29] KEPNER R.L. & PRATT J.R., 1994- Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiological, 58: 603-615. 137 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 CHANGES IN BACTERIAL ABUNDANCE AND BIOMASS IN SANDY SEDIMENT MICROCOSMS SUPPLEMENTED WITH GASOLINE Dan Răzvan POPOVICIU1, Ioan ARDELEAN1,2 Ovidius University of Constanţa,Natural Sciences and Agricultural Sciences Faculty, Mamaia Avenue, no. 124, Constanţa, 900527, Romania, e-mail: dr_popoviciu@yahoo.com 2 Biology Institute of Bucharest, Splaiul Independenţei, no. 296, 060031 Bucureşti, Romania, email:ioan.ardelean57@yahoo.com __________________________________________________________________________________________ 1 Abstract: Bacterial abundance, biomass and morphological diversity were studied in three marine sediment microcosms: control, sediment supplemented with gasoline, and sediment supplemented with gasoline and ammonium nitrate. Microbial density (4.7-6.65 × 106 cells/cm3 sediment in uncontaminated samples) and biomass (1.72-3.13 µg/cm3 sediment) dropped significantly after gasoline addition. Ammonium nitrate favoured a faster recovery to initial values. Gasoline contamination also modified the proportion of bacterial morphotypes, increasing the percentage of rod-shaped cells. Keywords: Bacteria, microcosms, hydrocarbons, abundance, biomass, morphotypes, sandy sediments. __________________________________________________________________________________________ 1. Introduction Hydrocarbon contamination is one of the most frequent and most dangerous forms of pollution affecting marine environments. Studying its effects on prokaryote communities is important, both theoretically and practically, opening the way to bioremediation. From a microbiological point of view, sediments and not the water column are the richest marine environment. Both sandy and muddy sediments show significant amounts of prokaryotes, playing a key role in the decomposition of organic matter and nutrient recycling.. Abundance, biomass and composition of sediment bacterial communities can be determined by many factors, such as the granulometric characteristics of the sediment, water dynamism, oxygenation, protozoan grazing etc [1, 2, 3]. Even though they cover a large part of the marine littoral enviroment, coastal sands are the less studied [1]. In order to evaluate the response of bacteriobenthos to various environmental changes, microcosms represent extremely valuable tools [4, 5]. The objective of the present study was to determine the effects of hydrocarbon addition on the ISSN-1453-1267 abundance, biomass and morphological diversity of bacteria in marine sandy sediment microcosms. 2. Material and Methods Microcosms. Sandy sediment was collected from the mediolittoral of a sandy beach in Constanţa, relatively close to the central headquarters of the “Ovidius” University, and wet sieved through a 2 mm sieve (in order to eliminate large particles and macrofauna) [6]. Three 1.4 L transparent plastic recipients were filled each with around 500 cm3 of sand, covered by a 200 mL sea water column. All the microcosms were covered with transparent caps and stored at constant temperature (18°C) with illumination simulating the day-night cycle. The control microcosm was labeled “A”. Microcosm B was supplemented with 95 gasoline (1% final concentration). Microcosm C was supplemented with the same amount of gasoline, plus ammonium nitrate as a nutrient (0.005% final concentration). Sampling and fixation. Five samples consisting of sediment cores were collected from each microcosm at time intervals of 14 days. The first series of cores was taken just before the addition of gasoline and nutrient. © 2010 Ovidius University Press Changes in bacterial abundance and biomass... / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010) Sample collection was done using improvised piston corers (20 mL syringes with the forepart detached, but with the gradation intact). From each sample, the surficial 5 cm3 (corresponding to a depth of 17.5 mm) was taken for analysis. Each sample was suspended in 5 ml of buffered formaline (4% final concentration) [7, 8, 9, 10]. The formaldehyde solution acts as a fixative, killing the microorganisms and preventing contamination and cell deformation. The labeled tubes containing the subsamples were preserved by refrigeration at +4°C. Cell separation. Dislodgement of bacteria attached to sand grains is an important step prior to analysis. The procedure used was adapted, with some modifications, from existing literature [11, 12, 13, 14, 15]. Sediment suspensions were diluted 5-fold, incubated with Tween 80 (1 mg/mL final concentration) for 15 minutes and vortexed at 2 400 r.p.m. for 5 minutes. Direct counting of bacteria. Microorganisms were visualised by epifluorescence microscopy, using 3,6-dimethylaminoacridinic chloride (acridine orange) as a fluorochrome. This compound becomes highly fluorescent by binding to the nucleic acids, giving an orange-red fluorescence for single-stranded nucleic acids (mostly RNA) and a green one for double-stranded acids (DNA) [16]. Acridine orange stains both living and dead cells [17, 18]. The technique employed was an adapted and simplified version of the protocols used by other authors [3, 8, 10, 11, 19, 20]. 1 ml was collected from each suspension and incubated for 5 minutes with 1 ml acridine orange (5 µg/mL final concentration). The resulting solution was filtered through a 0.45 µm Millipore filtering membrane, using a syringe and a Millipore holder. Filtered membranes were previously stained with Sudan Black, in order to reduce background fluorescence. Each filter was washed with 50-60 ml of distilled water, placed on a glass slide and examined using a Hund Wetzlar H 600 AFL 50 microscope, at an 500× enlargement. An eyepiece grid micrometer was employed. For each filter, 15-20 grids were randomly chosen (from different areas of the membrane, except for its margins), photographed with a digital camera and visualised with MBF ImageJ for Microscopy software (http://www.macbiophotonics.ca/downloads. htm.) [21]. Fluorescent cells in each grid were counted manually. Fluorescent anorganic particles and obviously eukaryotic structures (by size and morphology) were excluded. In case sediment particles masked bacterial cells, any bacteria found on the surface of such particles were counted twice [7, 8, 17, 22]. The mean bacterial density was calculated for each sample according to the following formula: N = n ×A f / A g × V / v where: N = mean bacterial number per cm3 of sediment; n = mean bacterial number per grid for each subsample; A g = grid area; A f = filter area; v = volume of the filtered sediment suspension; V = volume of the total sediment suspension containing 1 cm3 of sediment. Bacterial biomass estimation. All the microorganisms observed were classified into three morphological categories: cocci, bacilli (including coccobacilli and vibrios) and filamentous bacteria (those having a length more than five times greater than the width) [3]. Cell dimensions (diameter, respectively length and width) were measured using the grid micrometer. Biovolume was determined for each cell according to the formula [8, 16]: V = (π/ 4) d2 (l – d / 3) where: l = cell length; d = cell width/diameter. For cocci, the formula becomes: V = πd3 / 6 To determine dry biomass based on the biovolume, several authors proposed different conversion factors. In the present study, the following formula was used [23]: m d = 435 × V0,86 where: m d = dry biomass (fg); 140 Dan Răzvan Popoviciu, Ioan Ardelean / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010) V = cell volume (µm3). Dry biomass was determined for each cell, calculating then the media for each sample. Total (wet) biomass can be approximated using a conventional mean value for bacterial cell density, of 1.1 g/cm3 [23, 24]. cells/cm3, fine sands in a Mexican tropical lagoon, 1.2 m depth [27], 7 × 108-6.7 × 109 cells/cm3, Baltic Sea [28], over 5.12 × 108 cells/g dry sediment, Western Mediterranean Sea [29], 1.5 × 108 cells/g dry sand [10], 6-8 × 109 cells/g sediment [30] and 3.54-8.08 × 109 cells/g [3] in the Adriatic Sea, at several meters depth, 0.2-1 × 109 cells/g dry sediment, in littoral sands in the Gulf of Tokyo [14], 2.56-4.46 × 106 cells/g, at 2 m depth, in North Sea [31]. The addition of gasoline caused a decrease in cell abundance to values as low as 3.6 × 106 cells/cm3. A return to densities similar to the initial ones was observed in the last samples. The recovery was faster in the microcosm supplemented with ammonium nitrate (28 days). Direct cuantification of bacteria through epifluorescence microscopy has some limitations. Cell masking by sediment particles, background fluorescence, lack of an efficient method to distinguish prokaryotes from eukaryotes, the poor quality of some photographs etc., can cause overestimation or underestimation of real abundance [8, 17, 22]. The method used for bacterial dispersion from sediment grains can also influence the results [9]. An important factor that can cause underestimation of bacterial abundance is the extremely small size of some microorganisms. Many bacteria have diameters below 0.3 microns, and can be very difficult or even impossible to visualise, depending on the optical means employed. Some of them can even pass through usual filtering membranes. According to some authors such ultramicrobacteria constitute up to 72% of the soil microbiota, and it seems that they have similar proportions in marine environments [17]. In conclusion, all data obtained using direct counts should be regarded as relative. It should be noted that not all the bacteria ennumerated with acridine orange are alive. Living bacteria constitute usually less than one third, rarely reaching 60% of the total number. The rest are dead cells, or even cell fragments [10, 32]. 3. Results and Discussions Bacterial cell abundance. The evolution of cell density in time (from 0 to 56 days) for each microcosm is shown in Fig. 1. 7 6.5 Million cells 6 5.5 A 5 B 4.5 C 4 3.5 3 0 14 28 42 56 Tim e (days) Fig. 1. Number of bacterial cells (× 106) per cm3 of sediment. For undisturbed sediment cores, bacterial density ranged between 4.7-6.65 × 106 cells/cm3 sediment with an average of 5.52 × 106 cells/cm3. These values are within the variation limits of littoral sediment microbial density (although data found in literature is distributed over a wide range). For comparison, here are some bacterial densities: 109 cells/g dry sand [20], 5 × 108-1.5 × 109 cells/g sediment [9] and 7-9 × 107 cells/g [25] on the U.S.A. East Coast, 1.91-7.32 × 107 cells/g dry sediment, in Eastern Canada, at the waterline [1], 3.6 × 108 cells/g dry sediment, in Florida [26], 6.8-20.3 × 108 141 Changes in bacterial abundance and biomass... / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010) 3.5 Microcosm A 100% Biomass (µg) 3 80% A 2.5 2 Filamentous 60% B Rods C 40% Cocci 1.5 20% 1 0 14 28 42 0% 56 0 Tim e (days) 14 28 42 Tim e (days) 56 Microcosm B Fig. 2. Bacterial dry biomass (µg/cm3). 100% Bacterial biomass 80% Biomass showed large variations, from 1.36 to 3.13 µg/cm3 sediment (equivalent to 4.3 to 11.4 µg total biomass/cm3). On average, the highest biomass was determined for the undisturbed sediment (an average of 2.27 µg/cm3). The addition of gasoline was followed by a decrease in microcosms B and C. The average value for contaminated sediment in microcosm B was only 1.7 µg/cm3, while in C, it was higher (2.12 µg/cm3), showing a faster recovery. The importance of nitrogenous nutrients in the recovery of natural microbiota after hydrocarbon pollution is consistent with data in existing literature [33, 34]. The exact determination of bacterial biomass can be affected by various technical and mathematical factors. Different fluorochromes can give different results [16]. The selected biovolume to biomass conversion factor influences the final results. Also, it was demonstrated that coastal marine sediments contain significant numbers of disk-shaped bacteria and counting them as cocci would overestimate their volume [35]. Proportion of major bacterial morphotypes. As specified above, bacteria were classified into three groups: cocci, rods and filamentous. Their proportion in the total abundance, for each microcosm and collection time is shown in Fig. 3 (a,b,c). Filamentous 60% Rods Cocci 40% 20% 0% 0 14 28 42 Tim e (days) 56 Microcosm C 100% 80% Filamentous 60% Rods Cocci 40% 20% 0% 0 14 28 42 Tim e (days) 56 Fig.3 (a,b,c). Percentage of major bacterial morphotypes 142 Dan Răzvan Popoviciu, Ioan Ardelean / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010) Most of the bacterial cells (72-92%) observed were spherical (in accordance to data obtained in marine sediments by Šestanović et al. [3] and Popoviciu [36]). The proportion of rod-shaped bacteria (including coccobacilli and vibrios) was different among the three microcosms. In undisturbed sediment, their percentage was generally below 15% (note: in microcosm A, at T3, the high percentage was due to a single large colony of small rods), with an average of 13.9%. In gasoline contaminated sediment, rodshaped bacteria constituted a larger part of the microbiota, with an average of 23.4%. The proportion of filamentous bacteria was insignificant. Bacterial assemblages were rare, in concordance to the observations made by Novitsky & MacSween [1]. It should be noted that classification of small bacteria (cells with diameters below 0.6 microns formed the majority) into morphotypes is prone to errors. This is due to the fluorescent halo that appears around cells, causing very small sized bacilli or vibrios to be counted as cocci [19]. a) a) 4. Conclusions Hydrocarbon contamination affects marine sediment microbiota in terms of abundance, biomass and composition. Addition of nitrogenous nutrients (ammonium nitrate) favours a faster recovery to initial parameters. Epifluorescence microscopy is a useful tool for evaluating the reaction of sediment bacteria to environmental changes. In perspective, use of differential fluorochromes and correlation to cultivation techniques are to be employed in such studies. b) 5. References [1] NOVITSKY, J.A., MACSWEEN, M.C., 1989 – Microbiology of a high energy beach sediment: evidence for an active and growing community. Mar. Ecol. Prog. Ser. 52: 71-75. [2] BRUNE, A., FRENZEL, P., CYPIONKA, H., 2000 – Life at the oxic-anoxic interface: microbial activities and adaptations. FEMS Microbiol. Rev. 24: 691-710. c) Fig. 4. Main bacterial morphotypes: a) cocci; b) rods; c) filamentous (bar = 10 µm) 143 Changes in bacterial abundance and biomass... / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010) [3] ŠESTANOVIĆ, S., SOLIĆ, M., KRUSTULOVIĆ, N., BOGNER, D., 2005 – [4] [5] [6] [7] [8] [9] [10] [11] specific measures agree with whole-system metabolism? Mar. Ecol. Prog. Ser. 11: 119-127. Volume, abundance and biomass of sediment bacteria in the eastern mid Adriatic Sea. Acta Adriat. 46: 177-191. RÖLING, W.F.M., MILNER, M.G., JONES, D.M., LEE, K., DANIEL, F., SWANNELL, R.J.P., HEAD, I.M., 2002 – Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation. Appl. Environ. Microbiol. 68: p. 5537-5548. MIRALLES, G., NÉRINI, D., MANTÉ, C., ACQUAVIVA, M., DOUMENQ, P., MICHOTEY, V., NAZARET, S., BERTRAND, J.C., CUNY, P., 2007 – Effects of spilled oil on bacterial communities of Mediterranean coastal anoxic sediments chronically subjected to oil hydrocarbon contamination. Microb. Ecol. 54: 646-661. FISH, K.M., PRINCIPE, J.M. 1994 – Biotransformations of Aroclor 1242 in Hudson River test tube microcosms. Appl. Environ. Microbiol. 60: 4289-4296. SCHALLENBERG, M., KALFF, J., RASMUSSEN, J.B., 1989 – Solutions to problems in enumerating sediment bacteria by direct counts. Appl. Environ. Microbiol. 55: 1214-1219. FRY, J.C., 1990 – Direct methods and biomass estimation. Meth. Microbiol. 22: 41-85. EPSTEIN, S.S., ALEXANDER, D., COSMAN, K., DOMPÉ, A., GALLAGHER, S., JARSOBSKI, J., LANING, E., MARTINEZ, R., PANASIK, G., PELUSO, C., RUNDE, R., TIMMER, E., 1997 – Enumeration of sandy sediment bacteria: Are the counts quantitative or relative? Mar. Ecol. Prog. Ser. 151: 11-16. LUNA, G.M., MANINI, E., DANOVARO, R., 2002 – Large fraction of dead and inactive bacteria in coastal marine sediments: comparison of protocols for determination and Appl. Environ. ecological significance. Microbiol. 68: 3509-3513. FALLON, R.D., NEWELL, S.Y., HOPKINSON, C.S., 1983 – Bacterial production in marine sediments: will cell- [12] ELLERY, W.N., SCHLEYER, M.H., 1984 – Comparison of homogenization and ultrasonication as techniques in extracting attached sedimentary bacteria. Mar. Ecol. Prog. Ser. 15: 247-250. [13] EPSTEIN, S.S., ROSSEL, J., 1995 – Enumeration of sandy sediment bacteria: search for optimal protocol. Mar. Ecol. Prog. Ser 117: 289-298. [14] KUWAE, T., HOSOKAWA, Y., 1999 – Determination of abundance and biovolume of bacteria in sediments by dual staining with 4’,6diamidino-2-phenylindole and acridine orange: relationship to dispersion treatment and sediment characteristics. Appl. Environ. Microbiol. 65: 3407-3412. [15] BENNETT, P.C., ENGEL, A.S., ROBERTS, J.A., 2006 – Counting and imaging bacteria on mineral surfaces. in Patricia, J., Maurice, A., Warren, L.A. (eds.). Methods of Investigating Microbial-Mineral Interactions. CMS Workshop Lectures, Vol. 14: 37-78, The Clay Mineral Society, Chantilly. [16] SHERR, B., SHERR, E., DEL GIORGIO, P., 2001 – Enumeration of total and highly active bacteria. Meth. Microbiol. 30: 129-159. [17] KEPNER, R.L., PRATT, J.R., 1994 – Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiol. Rev. 58: 603-615. [18] MCFETERS, G.A., YU, F.P., PYLE, B.H., STEWART, P.S., 1995 – Physiological assessment of bacteria using fluorochromes. J. Microbiol. Meth. 21: 1-13. [19] WATSON, S.W., NOVITSKY, T.J., QUINBY, H.L., VALOIS, F.W., 1977 – Determination of bacterial number and biomass in the marine environment. Appl. Environ. Microbiol. 33: 940-946. [20] MONTAGNA, P.A., 1982 – Sampling design and enumeration statistics for bacteria extracted from marine sediments. Appl. Environ. Microbiol. 43: 1366-1372. [21] COLLINS, T.J., 2007 – ImageJ for microscopy. BioTechniques 43: 25-30. 144 Dan Răzvan Popoviciu, Ioan Ardelean / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010) [22] GOUGH, H.L., STAHL, D.A., 2003 – Optimization of direct cell counting in sediment. J. Microbiol. Meth. 52: 39-46. [31] LUNAU, M., LEMKE, A., WALTHER, K., 2005 – An improved method for counting bacteria from sediments and turbid [23] LOFERER-KRÖßBACHER, M., KLIMA, J., PSENNER, R., 1998 – Determination of bacterial cell dry mass by transmission electron microscopy and densitometric image analysis. Appl. Environ. Microbiol. 64: 688-694. [24] BAKKEN, L.R., OLSEN, R.A., 1983 – Buoyant densities and dry-matter contents of microorganisms: conversion of a measured biovolume into biomass. Appl. Environ. Microbiol. 45: 1188-1195. [25] HYMEL, S.N., PLANTE, C.J., 1998 – Improved method of bacterial enumeration in sandy and deposit-feeder gut sediments using the fluorescent stain 4,6-diamidino-2phenylindole (DAPI). Mar. Ecol. Prog. Ser. 173: 299-304. [26] PROCTOR, L.M., SOUZA, A.C., 2001 – Method for enumeration of 5-cyano-3,2-ditoyl tetrazolium chloride (CTC)- active cells and cell-specific CTC activity of benthic bacteria in riverine, estuarine and coastal sediments. J. Microbiol. Meth. 43: 213-222. [27] FERRARA-GUERRERO, M.J., CASTELLANOS-PAÉZ, M.E., GARZAMOURIÑO, G., 2007 – Variation of a benthic heterotrophic bacteria community with different respiratory metabolisms in Coyuca de Benitez coastal lagoon (Guerrero, Mexico). Rev. Biol. Trop. (Int. J. Trop. Biol.) 55: 157-169. [28] DIETRICH, D., ARNDT, H., 2000 – Biomass partitioning of benthic microbes in a Baltic inlet: relationships between bacteria, algae, heterotrophic flagellates and ciliates. Mar. Biol. 136: 309-322. [29] DANOVARO, R., FABIANO, M., BOYER, M., 1994 – Seasonal changes of benthic bacteria in a seagrass bed (Posidonia oceanica) of the Ligurian Sea in relation to origin, composition and fate of the sediment organic matter. Mar. Biol. 119: 489-500. [30] PUSCEDDU, A., FIORDELMONDO, C., DANOVARO, R., 2005 – Sediment resuspension effects on the benthic microbial loop in experimental microcosms. Microb. Ecol. 50: 602-613. environments by epifluorescence microscopy. Environ. Microbiol. 7: 961-968. ZWEIFEL, U.L., HAGSTRÖM, Å., 1995 – Total counts of marine bacteria include a large fraction of non-nucleoid-containing bacteria (ghosts). Appl. Environ. Microbiol. 61: 21802185. HIGASHIHARA, T., SATO, A., SIMIDU, U., 1978 – An MPN method for the enumeration of marine hydrocarbon degrading bacteria. Bull. Japan. Soc. Sci. Fish. 44: 1127-1134. HAZEN, T.C., 2010 – Biostimulation, in Timmis, K.N. (ed.). Handbook of Hydrocarbon and Lipid Microbiology, 4517-4530, SpringerVerlag Berlin Heidelberg. MUDRYK, Z.J., PODGÓRSKA, B., 2006 – Scanning electron microscopy investigation of bacterial colonization of marine beach sand grains. Baltic Coastal Zone 10: 61-72. POPOVICIU, D.R., 2009 – Aspecte cantitative şi morfo-structurale ale microbiotei din sedimente marine nisipoase de la litoralul românesc al Mării Negre. Master’s thesis, Ovidius University of Constanţa, Faculty of Natural Sciences and Agricultural Sciences. [32] [33] [34] [35] [36] 145 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 THE FORMATION OF BACTERIAL BIOFILMS ON THE HYDROPHILE SURFACE OF GLASS IN LABORATORY STATIC CONDITIONS: THE EFFECT OF TEMPERATURE AND SALINITY Aurelia Manuela MOLDOVEANU *, Ioan I. ARDELEAN ** * Ovidius” University Constanta, 1Universitatii Alley, Building B, 900527 Constanta, Romania, aurelia.moldoveanu@yahoo.com ** Biology Institute of Bucharest, 296 Splaiul Independenţei, 060031 Bucharest, Romania, ioan.ardelean57@yahoo.com __________________________________________________________________________________________ Abstract: In the case of temperature variation, at 18ºC there is an increase of the cellular density from 12∙102cel/mm2 to 62∙102cel/mm2, while at 6 ºC cellular density increases from 5∙102cel/mm2 to 55∙102 cel/mm2. The results obtained show that cellular density in the case of biofilms formed at 6 ºC is lower compared to cellular density of biofilms formed at 18 ºC. Salinity modification from 15g/l to 10g/l determined an increase of cellular density from 4∙102 cel/mm2 to 54∙102 cel/mm2, while the modifications of the osmotic conditions in the marine environment due to salinity decrease to 5g/l led to an increase of the cellular density from 2∙102 cel/mm2 to 49∙102 cel/mm2. The variation of temperature and salinity of seawater in “in vitro” conditions influenced the process of bacterial adherence and formation of the initial layers of the biofilms by the modification of the density of the adherent cells. Keywords: quorum sensing, exopolysaccharides, matrix, microecosystem, microfouling. __________________________________________________________________________________________ 1. Introduction Biofilms are complex structures made up of cells and exopolysaccharides which form at the level of interfaces and which are intensely studied because of their fundamental importance and applicability in the environmental domain, biotechnology and medicine [1,2,3,4,5]. Marine bacteria form biofilms in “in situ” conditions, under the influence of various environmental factors. Hydrostatic pressure, solar radiation, temperature, salinity, pH, oxidation potential and nutrients existing on the surfaces are physic-chemical factors that influence the activity of microorganisms, but their role on the marine bacterial populations is still being studied [6,7,5]. Among these factors, temperature and salinity have major importance for all living organisms, especially for those in the marine environment, where microorganisms are subjected to extremely wide variations which allowed them to survive from the beginning of life on Earth. They are the only ISSN-1453-1267 organisms that can adapt to extreme environments [8,9,10]. In laboratory conditions, the variation of environmental factors is essential in the formation of biofilms. It is important to know which factor has the most influence on the adherent bacterial cells. Thus, the growth and multiplication of microorganisms is the result of a number of coordinated metabolic reactions whose normal development is ensured by an optimal temperature [11,12,13]. The marine bacteria in the structure of biofilms react to temperature and modify their bacterial metabolism and the mechanism for the regulation of genes depending on how this factor varies, most species being studied between their optimal temperature limits due to the mesophile character [14]. Thus, an increase by 10 ºC of the initial temperature determines an increase of the speed of the chemical reactions and gene regulation mechanism. Consequently, the speed of the enzymatic processes increases progressively as the temperature © 2010 Ovidius University Press The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) rises until it reaches the optimum level and then the speed decreases progressively [15]. The natural environments offer microorganisms different conditions of salinity, from very low concentrations (rivers and lakes) to the very high concentrations of salty lakes and seas, or even to those that represent true saturated solutions. Thus, salinity becomes a variable factor in the marine environment which is important for the microorganisms within the biofilms as they are influenced by it according to the degree of tolerance to the concentration of NaCl and the mechanism for the regulation of the available ions [16]. The study of the effect of temperature and salinity on the temporal dynamics of bacterial cell density on the hydrophile surface of glass in laboratory static conditions leads to personal data regarding the initial stages of biofilm formation. water recirculation, according to [24], who claims that the methods with continuous flux prevent the rapid formation of biofilms within the first hours. The support slides for the adherent bacteria were positioned according to the mentioned methods in an inclined position compared to the classical method, in order to avoid the sedimentation phenomenon which determines the occurrence of high densities of the adherent marine bacteria [25]. The experiment was accomplished in a thermostatic room at a constant temperature of 18 ºC in the Laboratory for Biodiversity Investigation within “Ovidius” University of Constanta and in a refrigerator at a constant temperature of 6 ºC. The salinity modification was done only for the littoral seawater and not for the aquarium water which is a microecosystem. Thus, seawater salinity, which has a normal value of 15g/l was modified by adding osmosis water and certain mixtures per liter obtaining thus two experimental versions: in the first version, normal salinity was decreased to 10g/l by adding 333ml of osmosis water in 666ml of seawater; in the second version a salinity of 5g/l was obtained by adding 666 ml of osmosis water in 333 ml of seawater. The study of biofilms was accomplished over a period of 36 hours during which there was an interval when no samples were collected. Sample collection occurred for 12 hours in the first day hourly, followed by an interval of 12 hours when no samples were collected and again the following day samples were collected every two hours for 12 hours. After collection the slides were subjected to a process of fixation with 2.5% formaldehyde solution in artificial seawater (solution with marine salts with a concentration of 18g/l, similar to the Black Sea) for 30 minutes and then subjected to desalinization by washing for 10 minutes in three successive solutions with the following content: 75% artificial seawater with 25% osmosis water, 50% artificial seawater with 50% osmosis water and 100% osmosis water. The desalinization was realized in order to prevent the formation of salt crystals which absorb the fluorescent coloring matter and reflect it, affecting thus the cell visualization [26]. After desalinization, the samples were introduced in a solution with 0.5% gentian violet in 10 ml ethanol and 90 ml distilled water for one 2. Material and Methods In our experiments, we used two static methods in order to determine the environmental factors with role in biofilm formation: the Henrici method [17,18,19], widely employed in the study of adherence and the microbial fishing method [20,21], a more recent adaptation of the classical method. The surfaces were subjected to a sterilization process in order to diminish the possible contamination with microorganisms of the glass slides which will serve as support for the adherent marine bacteria. The slides were degreased with ethanol 70% and sterilized in the drying oven at 180 ºC for one hour [22]. In order to obtain biofilms, two types of liquid culture media were used: seawater from the littoral zone and seawater kept in aquarium conditions in the Laboratory for Biodiversity Investigation within “Ovidius” University of Constanta. The aquarium seawater is frequently used in the study of biofilms and marine microfouling and it was used in order to observe the possible facilitation of their formation [23]. The method used is accomplished in static conditions in sterile containers in which 100 ml of seawater were poured and the slides were introduced. This type of method is more advantageous for the formation of biofilms when there is no system for 148 Aurelia Manuela Moldoveanu, Ioan Ardelean / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) minute. Afterwards, they were abundantly washed twice with osmosis water in order to eliminate the excess of coloring matter [27]. The sample investigation was done by means of the Hund microscope, cell counting being done using an ocular grid, calibrated according to the standard procedure [28]; cells within 20 microscopic fields per each sample were counted, according to the standard counting procedures for the surface bacteria [29]. Thus, the values of cellular density are expressed on the graphs represented by the mean of the 80 microscopic fields per each sample. is enclosed in an extracellular polymeric substance matrix. 3.2 The formation of biofilms under the influence of temperature Figure one shows the values of cellular density obtained after the modification of the temperature factor for the biofilms formed on the hydrophile surface of glass slides and collected from the containers with littoral seawater kept at a constant temperature of 18ºC and 6 ºC, respectively. The data analysis emphasized the existence of successive stages for the formation of biofilms. Thus, in the case of the biofilms formed at 18 ºC, one hour after the slides immersion the cellular density is 12∙102 cel/mm2. This value doubles eight hours later to 25 ∙102 cel/mm 2 and increases progressively to a tendency to triple the cellular density to 37 ∙ 102 cel/mm 2 11 hours later. After 12 hours, during which the slides were left over night, the following day the cellular density reaches the value of 45∙102 cel/mm2 24 hours after immersion. The value increases progressively to 60∙102 cel/mm2 36 hours after immersion For the seawater in the containers kept at 6 ºC in the refrigerator, there is a progressive increase from 5 ∙102 cel/mm2 only one hour after immersion and a doubling of this value seven hours later to 10 ∙102 cel/mm2, as well as tripling to 15∙102 cel/mm2 eight hours later. The following day, after 12 hours, the cellular density was 41∙102 cel/mm2 and increased progressively to 54∙102 cel/mm2 . The progression of cellular density growth is over 2.3 for the biofilms formed at 18 ºC during the first 12 hours and below 1.2 after 24 hours. Also, in the case of the biofilms formed in containers kept at 6 ºC, the progression is over 1.9 during the first 12 hours and below 1.1 after 24 hours. On the first day, after 12 hours, there is a difference of approx. 9∙102 cel/mm2 between the two progressions of density growth, depending on temperature. 24 hours later, the difference is below 8∙102 cel/mm2 and 36 hours later it rises to 10∙102 cel/mm2. A number of experiments regarding bacterial adhesion were accomplished on different types of surfaces (copper, PVC and polybuten) by Rogers [31] at different temperatures (20 ºC, 40 ºC, 50 ºC and 60 3. Results and Discussions 3.1 The chemical analysis of water There are differences between the two types of culture media used for the generation of bacterial biofilms on the hydrophile surface of glass slides and in order to emphasize their existence we analyzed the seawater samples in the Chemistry Laboratory within the “Grigore Antipa” Marine Research Institute of Constanta (Table 1). The chemical analysis of seawater emphasized the existence of differences among the chemical parameters: salinity, pH, concentration of inorganic substances between the two types of seawater used. The littoral seawater has normal parameters also registered in previous years [30], but the aquarium seawater has values well over the normal limit with an increase of over 10g/l of salinity and a decrease of pH from 8.12 (normal value for littoral seawater) to 6.56 units for the aquarium water, almost two units less than the initial value. The concentration of inorganic substances is well above the normal one for seawater. The concentration of nitrates is three times higher compared to the normal value, while the concentration of polyphosphates is over 84 times higher. The existence of these differences between the two used culture media can cause changes in the formation manner of bacterial biofilms in liquid medium, as well as the temporal dynamic of their formation. The bacterial biofilms formed are an assemblage of surface-associated microbial cells that 149 The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) ºC) using the species Legionella pneumophila (a species with wide temperature limits between 5.7 and 63 ºC) and other strains of non-Legionella type over a period of 21 days. As a result, it was observed that the used strains displayed a logarithmic growth with a density value of 1.3∙104cel/cm 2 in the growing phase and 7.56∙104cel/cm 2 on polybutylene and PVC surfaces for the non-Legionella strains at 20 ºC and 4.25∙104cel/cm 2 for polybutylene surface at 60 ºC. It is evident that the colonization is higher on the hydrophobe surfaces at 20 ºC, compared to 60 ºC when the number of bacteria decreases due to the exceeding of the optimal temperature for microorganism development. Experiments regarding the colonization of surfaces by the bacterium Bdellovibrio bacteriovorus were accomplished by Kelley [32] under the influence of different temperatures (between 4 and 29 ºC), using clam valves, glass and polystyrene as substrate, and observing the existence of positive correlations in the case of the factor temperature and the formation of biofilms, with maximum association of cells in the biofilms at 18 ºC and a minimum one at 14 ºC, as well as a significant decrease of density at temperatures below 5 ºC after 24 hours, followed by a progressive increase of density 120 hours after the beginning of the experiment. The values obtained demonstrated a logarithmic increase of the number of adherent cells from 1.1 ∙105 CFU/cm 2 to 1.4∙105 CFU/cm 2 for the clam valves, 1.7∙103 CFU/cm 2 and 1.8∙104 CFU/cm 2 for glass and 5.4∙103 CFU/cm 2 and 1.0 ∙104 CFU/cm 2 for polystyrene. A number of experiments regarding the capacity of accomplished certain isolates of Stenotrophomonas maltophilia to form biofilms in variable temperature conditions (18 ºC, 32 ºC, 37 ºC) by were realized by Di Bonaventura [33] using different strains. There is an increase of the quantity of biofilms for the strains exposed to 32 ºC after one day to 0.680 BPI compared to those exposed to 18 ºC (0.557 BPI) and 37 ºC (0.491 BPI). In what regards the used strains, the temperature did not modify significantly their distribution: 82% of those used formed biofilms and only 2% did not form them. One strain formed biofilms only at 18 ºC and two strains only at 32 ºC. The capacity to forms biofilms is important even at room temperature (18 ºC), but the adherence value is lower. The following day, after the 12 hour interval when no samples were collected, the density value was 41∙102 cel/mm 2 and there was a progressive growth towards 55∙102 cel/mm2 . Data in specialized literature confirm the existence of a growth in bacterial density depending on the exposure time of the surfaces to aquatic environment and the increase of temperature. Thus, at 18 ºC, up to 25-30 ºC, there is an optimal bacterial growth. But temperatures over 35 ºC, 40 ºC and 50 ºC affect the formation of biofilms because the optimum limits for the survival of certain bacterial species are exceeded Our experiments took place between the optimum limits for mesophile bacteria, noticing an increase of the density values at 18 ºC, compared to 6 ºC (kept in a refrigerator). In the case of the slides immersed in containers with aquarium water at 18 ºC, Figure 2 displays an increase of the bacterial density from 16∙102 cel/mm 2 one hour after immersion to a double value of 32∙102 cel/mm 2 eight hours later. After 12 hours, during which the slides were left over night, there is a tendency for the tripling of the cellular density to 49∙102 cel/mm 2 22 hours after the immersion of the slides into liquid medium and a progressive increase towards 62∙102 cel/mm 2. For the containers kept at 6 ºC, the density increases to 5∙102 cel/mm 2 one hour from immersion towards a double value of 13∙102 cel/mm 2 seven hours later and a progressive growth from 22 ∙102 cel/mm 2 ten hours later when there is a tendency for a triple value of the density of adherent bacteria. The growth occurs based on a progression of 2.5 in the case of biofilms formed in aquarium water kept at 18 ºC during the first 12 hours. There is a decrease to 1.1 after 24 hours from immersion. The difference between the two progressions is 4∙102 cel/mm 2 during the first 12 hours from immersion and it increases to 8 ∙102 cel/mm 2 24 hours later. It decreases 36 hours later to 7 ∙102 cel/mm 2 . In variable conditions of temperature, the bacterial colonization occurs more quickly in aquarium seawater. Thus, Toren [34] realizes experiments regarding the formation of biofilms (Vibrio sp. strain AK-1) on a coral surface in case of 150 Aurelia Manuela Moldoveanu, Ioan Ardelean / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) temperature variation (16º C, 23º C, 29º C) and registers a decrease of the quantity of inoculate from 1.2 ∙108 cel/l to 1.2∙102 cel/l used with the increase of temperature, as well as an increased adhesion at high temperatures during the first hours from immersion. Experiments were realizes by Else [5] regarding the bacterial colonization of the hydrophile surface of metals (stainless steel, titanium and nickel) in variable conditions of temperature (30º C, 60º C and 70º C) and humidity over a longer period of time (from a few days to 18 months). They observed an increase of adherent bacteria between 1.06∙102 cel/cm 2 and 7.61 ∙102 cel/cm 2 at a temperature of 30º C on steel plates. They also observed a decrease of the number of bacteria from the first day for the plates exposed to high temperatures (60º C and 70º C), especially on those of nickel and steel. In what regards the role of the bacterial film in the mediation of invertebrate attachment and fouling formation, Lau et al. [35] realized experiments at different temperatures (16º C, 23º C and 30º C), noticing an increase in the number of bacteria from 14.3∙103 cel/mm -2 at 16º C to 21.2∙103 cel/mm -2 at 30º C. The experiments emphasized a more significant influence of the temperature on the biomass than on the bacterial density. A number of experiments regarding the formation of biofilms in different conditions of temperature were realized by Di Bonaventura [36] together with other collaborators accomplish in 2007 (4º C, 12º C, 22º C, 37º C) by Listeria monocytogenes on the hydrophile surface of glass, steel and the hydrophobe surface of polystyrene. The results emphasized a progressive increase on the surface at 4 ºC of 0.206, at 12 ºC BPI to 0.233 BPI, 22º C to 0.366 BPI, in comparison to polystyrene and stainless steel. At 37º C the values are close to those from the three surfaces studied, but there is also greater species variability. Still, the most considerable growth of 1.275 was obtained on the hydrophobe surface of polystyrene. Bacterial density registers an increase of the adherent bacteria with higher values for the biofilm formed in aquarium water kept at 18 ºC, compared to the one kept at 6 ºC. The values obtained are higher than those for seawater, which is due to the different physical and chemical properties of aquarium water and to the nutrients. Adherent marine bacteria attach themselves to surfaces and form microcolonies in the first hour after immersion in the marine medium. They grow in size with the immersion period, data confirmed by [24]. 3.3 The formation of biofilms under the influence of salinity Variation of salinity was done in order to observe the influence of osmotic conditions on the process of bacterial adherence and the formation of the initial phases of biofilms. For the slides immersed in containers with seawater with 15g/l salinity, Figure 3 displays an increase of bacterial density to 12∙102 cel/mm 2 one hour after immersion towards a doubling of this value to 25∙102 cel/mm 2 eight hours later. The slides were left over night for 12 hours and the following day there was a progressive increase of the cellular density value of 49∙102 cel/mm 2 where there is a tendency for a triple value towards 62∙102 cel/mm2. In the case of containers with seawater with modified salinity (addition of osmosis water 10g/l), the bacterial density increased to 4∙102 cel/mm 2 one hour after immersion to a double value of 8∙102 cel/mm 2 after four hours and the progressive increase from 18 ∙102 cel/mm 2 after eight hours when there is a tendency to triple the value of bacterial density. After the 12 hour interval when the slides were left over night in containers, there is an increase of the cellular density from 41∙102cel/mm2 24 hours after immersion to 54∙102 cel/mm 2 36 hours after immersion. The growth progression during the first 12 hours in the case of the biofilms formed at a salinity of 15g/l is higher, with a value of 2.3 and displays a decrease after 24 hours to 1.1. In the case of the biofilms formed at a salinity of 10g/l, the growth progression is 2.4 during the first 12 hours and it decreases after 24 hours to 1.1. The difference between the two progressions is 2∙102 cel/mm 2 in the first 12 hours, it increases to 8∙102 cel/mm 2 24 hours after immersion and remains constant at this value until 36 hours. In the case of containers with seawater with modified salinity (addition of osmosis water 5g/l), Figure 4 displays an increase of bacterial density from 2∙102 cel/mm 2 one hour after immersion to a 151 The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) double value of 4∙102 cel/mm 2 three hours later and the progressive increase to 9 ∙102 cel/mm 2 six hours later when the tendency is for a triple value of the bacterial density. The slides collected after 12 hours display a progressive increase of cellular density from 31∙102cel/mm2 24 hours after immersion to 49∙102 cel/mm 2 36 hours later. The increase is accomplished based on a progression with a value of 2.3 in the first 12 hours in the case of the biofilms formed at 15g/l salinity and a decrease of this value to 1.1 after 24 hours. For the biofilms formed at 5g/l salinity, the value of the growth progression is 1.5 in the firs 12 hours, which drops to 1.4 in the following 24 hours. Between the two growth progressions there are differences between the values of cellular density. Thus, after 12 hours, the difference is 13∙102 cel/mm 2 and it drops after 24 hours to 8∙102 cel/mm 2, but increases after 36 hours to 13∙102 cel/mm 2. While studying the colonization of surfaces by the bacterium Bdellovibrio bacteriovorus, [32] realized experiments under the influence of different temperatures and observed the existence of a colonization tendency and biofilm formation between 3.4 g/l and 35 g/l. Salinity influenced the formation of biofilms even at values below 5 g/l, the number of adherent bacteria in the biofilm formed at 11g/l salinity being well over the expected one. At 4g/l salinity there is a decrease in the number of cells from 3.5∙106 CFU/cm 2 to 3.8∙104 CFU/cm 2 five days after immersion. Some experiments regarding the role of bacterial biofilm were accomplished by [35] in the mediation of invertebrate attachment and microfouling formation at different temperatures and salinity values of 20g/l-34g/l. There is bacterial increase from 12.8∙103 cel/mm -2 to 20g/l la 21.2∙103 cel/mm -2 at 34 g/l. The experiments revealed no significant correlation between salinity and bacterial density in regards to biomass. Some experiments were accomplished in regards to the role of salinity (between 12g/l and 80g/l) in the surface corrosion [37] achieves some experiments using stainless steel as substrate. They revealed the existence of a drop of cellular density with the increase of water salinity, noticing a corrosion maximum at 35 g/l between 1.7 ∙109 CFU/cm2 and 2.1∙10 CFU/cm2 for the aerobe species analyzed. The experimental data have increased values compared to those obtained by our experiments. The values of cellular density emphasize an increase correlated with the modification of salinity value as a whole, salinity increase from 5g/l to 10g/l and to 15g/l, the normal average value for seawater. These data are confirmed by the specialty literature as long as the increase is recorded between certain optimum salinity limits. The density values obtained when salinity was modified to 5g/l are lower than those for salinity from 10g/l and 15g/l, which demonstrates that a possible supply of fresh water in the natural environment may influence the formation manner of the biofilms. Microcolonies form from the very first hours after the immersion of the hydrophile surfaces in the case of salinity variation as well. These data are confirmed by [24] in the specialized literature in the case of experiments for the formation of biofilms in static conditions. 4. Conclusions The environmental factor such as temperature and salinity seems to influence bacterial adherence. The formation of the initial layers of the biofilms and their temporal dynamics in “in vitro” conditions determines a progressive increase of cellular density and the formation of microcolonies from the first hour after immersion in liquid medium. The modification of temperature and salinity values determined a decrease of the total number of adherent cells, compared to the normal one on the hydrophile surface of glass, by mechanism(s) which are under investigation. 5. References [1] ZOBELL C.E., 1943 – The effect of solid surfaces upon bacterial, J. Bacteriol., 46 (1): 39– 56. [2] COSTERTON J.W., GEESEY G.G., CHENG K.J. 1978 – How Bacteria Stick, Scientific American; 238 (1): 86-95. [3] ZARNEA GH., 1994 – Tratat de Microbiologie generalã, Ecologia microrganismelor, Vol. 5, Ed.Academei Române, Bucureşti. 152 Aurelia Manuela Moldoveanu, Ioan Ardelean / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) [18] LEWANDOWSKI Z. AND STOODLEY P., [4] LAZAR V. 2003- Aderenţa microbianã, 1995 - Flow induced vibrations, drag force, and Academiei Române, Bucureşti , 216 pp. [5] ELSE T. A., PANTLE C. R., AMY P. S., 2003pressure drop in conduits covered with biofilm, Boundaries for Biofilm Formation: Humidity and International IAWQ Conference Workshop on Temperature, App. .and Environ. Microbiology, Biofilm Structure, Growth, and Dynamics, 69(8): 5006–5010. Noordwijkerhout, The Netherlands. [6] CARLUCCI A. F. AND PRAMER D., 1959[19] POSCH T., FRANZOI J., PRADER Factors Affecting the Survival of Bacteria in Sea M.,.SALCHER M., 2009 - New image analysis Water, Microbiological Process Report, 7: 388tool to study biomass andmorphotypes of three 392 major bacterioplankton groups in an alpine lake, [7] DUFOUR PH., 1982 - Influence des conditions Aquatic Microbial Ecology, 54 : 113–126. de milieu sur la biodéradation des matieres [20] COSTERTON J.W, GEESEY G.G, CHENG organiques dans une lagune tropicale K.J. - 1978 How Bacteria Stick, Scientific Oceanologica Acta, 5 ( 3): 355-363. American, 238 (1): 86-95 [8] ROSZAK D.B. AND WELL C., 1987 – Survival [21] http://www.BiofilmsONLINE.com, 2008: Strategies of Bacteria in the Natural Techniques & Protocols, Henrici’s Microbial Environement, Micro. Rev. 365-379. Capture Technique. [9] COSTERTON J.W. 2007- The biofilm Primer, [22] LAZAR V., HERLEA V., CERNAT R., Springer, 200 pp. BALOTESCU M., BULAI D., MORARU A., [10] PARSEK M. R. GREENBERG E.P., 2008 2004 - Microbiologie generala, Manual de Sociomicrobiology: the connections between lucrarii parctice, Universitatii Bucuresti, 320 pp. quorum sensing and biofilms Trends in Microbioy , [23] HOVANEC T. A. AND DELONG E. F., 199613 ( 1): 28-33. Comparative Analysis of Nitrifying Bacteria [11] ZOBELL C. E. AND CONN J. E., 1940 Associated with Freshwater and Marine Aquaria, Studies on the thermal sensitivity of marine Appl. Environ. Microbiol, 62 (8) : 2888–2896. bacteria, J. Bacteriol., 40 (2): 223–238. [24] MERRITT J. H., KADOURI D. E., AND. [12] MITTELMAN M.W.1998 - Structure and O’TOOLE G. A 2005- Growing and Analyzing Functional Characteristics of Bacterial Biofilms, Static Biofilms, Current Protocols in Dairy Sci., 81: 2760–2764. Microbiology, 1B.1.1-1B.1.17. [13] COMPERE C., 1999 - Biofilms en mileu marin, [25] KUMAN A. AND PRASAD R., 2006 - Biofilms Techniques Sciences Methodes, 11: 48-54. Jk. Science., 8 (1): 14 -17. [14] WHITE-ZIEGLER C. A., SUZIN U., PEREZ N. [26] RUBIO C., 2002 - Comprehension des M., BERNS A. L., MALHOWSKI A.J. AND mecanismes d’adhesion des biofilms en milieu YOUNG S., 2008- Low temperature (23ºC) marin en vue de la conception de nouveaux increases expression of biofilm, cold-shock- and motens de prevention, These de dcotorat, Paris,. RpoS-dependent genes in Escherichia coli K-12, 216 pp. Microbiology, 154 : 148–166. [27] SONAK S,BHOSLE N., 1995- A simple method [15] EDDLEMAN H., 1998 - Optimum Temperature to assess bacterial attachment to surfaces, for Growth of Bacteria, Indiana Biolab, 14045 Biofouling, 9(1): 31-38 Huff St., Palmyra IN 47164 [28] HULEA A 1969 - Ghid pentru labortoarele de [16] STANLEY S. 0. AND MORITA R. Y., 1968 micologie şi bacteriologie. Ed. Agrosilvică, Salinity Effect on the Maximal Growth Bucureşti. Temperature of Some Bacteria Isolated from [29] FRY, J.C., 1990 – Direct methods and biomass Marine Environments, J. of Bacteriology, 95 (1) estimation. Meth. Microbiol, 22: 41-85. : 169-173. [30] COCIASU A., LAZAR L. AND VASILIU D. [17] HENRICI, A.T., 1933 -Studies of Freshwater 2008 –New Tendency in nutrient evolution from Bacteria: I. A Direct Microscopic Technique. romanian costal waters, Cercetarii marine INCDM, 38: 7-23. 153 The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) [31] ROGERS J., DOWSETT A. B., DENNIS P. J., LEE J. V., AND KEEVIL C. W., 1994 Influence of Temperature and Plumbing Material Selection on Biofilm Formation and Growth of Legionella pneumophila in a Model Potable Water System Containing Complex Microbial Flora, Appl. Environ. Microbiol., 60 (5): 1585-1592. [32] KELLEY J. I., TURNG B.F, WILLIAMS H. N., AND BAER M. L., 1997 - Effects of Temperature, Salinity, and Substrate on the Colonization of Surfaces In Situ by Aquatic Bdellovibrios, Appl. Environ. Microbiol, 63: 8490. [33] DI BONAVENTURA G., STEPANOVIC S., PICCIANI C., POMPILIO A., PICCOLOMINI R., 2007- Effect of Enviromental Factors on Biofilm Formation by Clinical Stenotrophomonas maltophilia isolates, Folia Microbiol., 52 (1): 86-90. [34] TOREN L.A.., LANDAU L. KUSHMARO A., LOYA Y. AND ROSENBERG E., 1998 Effect of Temperature on Adhesion of Vibrio Strain AK-1 to Oculina patagonica and on Coral Bleaching , Appl. Environ. Microbiol, 64 (4): 1379–1384. [35] LAU S.C.K, THIYAGARAJAN V., CHEUNG S.C.K, QIAN P.Y., 2005 - Roles of bacterial community composition in biofilms as a mediator for larval settlement of three marine invertebrates, Aquat Microb. Ecol, 38: 41–51. [36] DI BONAVENTURA G., PICCOLOMINI R., PALUDI D., D’ORIO V., VERGARA A., CONTER M. AND IANIERI A., 2007Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity, J. Appl. Microbio., 104 : 1552–1561. [37] FRANCA F.P., FERREIRA C.A., LUTTERBACH M.T.S., 2000- Effect of different salinities of a dynamic water system on biofilm formation, J. of Industrial Micro. & Bioteh., 25: 45-48. 154 The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) Table 1. The values of the seawater chemical parameters (liquid culture medium) Chemical parameters Sea water (zona litorala) salinity pH P-PO 4 N-NO 2 N-NH 4 N-NO 3 Si-SiO 4 Sea water (Aquarium) 25.10 g/L 6.56 unit. 63.80 µmoli/dm3 12.51 µmoli/dm3 5.46 µmoli/dm3 30.25 µmoli/dm3 0.24 µmoli/dm3 15.10 g/L 8.12 unit. 0.74 µmoli/dm3 0.42 µmoli/dm3 1.13 µmoli/dm3 3.14 µmoli/dm3 21.16 µmoli/dm3 Early succesion of biofilm s in containers over a period of 12 hours T0=24 hours Early succesion of biofilm s in containers over a period of 12 hours T0=0 hours 66 30 y = 2.3022x + 6.7253 25 Slides Sea Water (Tem p.18ºC) 61 Slides Sea Water( Tem p.18ºC) 2 cel/mm2) Density (10 2 Slides Sea Water ( Tem p.6ºC) Slides Sea Water( Tem p.6ºC) 35 2 Density (10 cel/mm) 40 R2 = 0.9354 20 15 y = 1.9066x + 1.7912 R2 = 0.9668 10 56 y = 1.25x + 43.107 R2 = 0.9895 51 46 5 y = 1.1429x + 35.714 R2 = 0.9922 41 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 36 0 Time (hours) 2 4 6 8 10 Time (hours) 12 14 Fig.1. The formation of a biofilm under the influence of temperature in containers with littoral seawater Ealy succesion of biofilm s in containers over a period of 12 hours T0=0 hours Early succesion of biofilm s in containers over a period of 12 hours T0=24 hours 65 45 40 Slides Aquarium (Tem p.18ºC) 60 Slides Aquarium ( Tem p.18ºC) 2 35 2 Density (10 cel/mm2) Density (10 cel/mm) Slides Aquarium (Tem p.6ºC) Slides Aquarium (Tem p.6ºC) y = 2.5165x + 10.363 R2 = 0.8844 2 30 25 20 y = 2.6429x + 1.9121 R2 = 0.9883 15 10 y = 1.125x + 47.839 R2 = 0.9883 55 50 y = 1.2321x + 39.661 R2 = 0.9919 45 5 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 40 14 0 Time (hours) 2 4 6 8 Time (hours) 10 12 14 Fig.2. The formation of a biofilm under the influence of temperature in the containers with aquarium seawater 155 Aurelia Manuela Moldoveanu, Ioan Ardelean / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) Ealty succesion of biofilm s in containers over a peiod of 12 hours T0=0 hours 65 40 Slides Sea Water(Sal.15g/l) Slides Sea Water ( Sal.15g/l) 30 60 2 Density (10 cel/mm2) 2 Slides Sea Water(Sal.10g/l) Slides Sea Water(Sal.10g/l) 35 2 Density (10 cel/mm) Ealry succesion of biofilm s in containers over a period of 12 hours T0= 24 hours y = 2.3022x + 6.7253 R2 = 0.9354 25 20 y = 2.4341x - 0.2198 R2 = 0.9793 15 10 y = 1.125x + 47.839 R2 = 0.9883 55 50 y = 1.1607x + 39.446 R2 = 0.9816 45 5 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 40 0 Time(hours) 2 4 6 8 Time (hours) 10 12 14 Fig.3. The formation of a biofilm under the influence of salinity decrease (from 15g/l to 5 g/l) in the containers with littoral seawater Early succesion of biofilm s in containers over a period of 12 hours T0= 0 hours 69 40 Slides Sea Water ( Sal.5g/l) Slides Sea Water ( Sal. 5g/l) 35 64 Slides Sea Water (Sal.15g/l) 30 2 Density (10 cel/mm2) 2 Density (10 cel/mm2) Early succesion of biofilm in containers over a period of 12 hours T0=24 hours y = 2.3022x + 6.7253 R2 = 0.9354 25 20 15 10 y = 1.5385x - 0.2308 R2 = 0.9926 5 Slides Sea Water (Sal.15g/l) y = 1.125x + 47.839 R2 = 0.9883 59 54 49 44 y = 1.4286x + 28.714 39 R2 = 0.9627 34 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 29 0 Tim e(hours) 2 4 6 8 Time (hours) 10 12 14 Fig.4. Biofilm formation under the influence of salinity decrease (from 15g/l to 10g/l) in containers with littoral seawater 156 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 THE CLINICAL UTILITY OF ADITIONAL METHODS IN EFFUSIONS EVALUATION Ana Maria CRETU*, Mariana ASCHIE**, Diana BADIU**, Natalia ROSOIU*** *Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology, Mamaia Avenue, No. 124, Constanţa, 900552, Romania, e-mail: cretu_anamaria@yahoo.com ** Clinical Emergency Hospital of Constanta, Department of Pathology, Tomis Avenue, No. 145, 900591, Constanta ***Ovidius University of Constanţa, Medicine Faculty,Mamaia Avenue, No. 124, Constanţa, 900527, Romania ________________________________________________________________________________________ Abstract: Cells from reactive or hyperplasic mesothelium shed from body cavity surface, in various biological conditions, may present a wide range of deviation from normal cellular morphology, making it difficult, or even impossible, to distinguish them from malignant cells by mean of purely cytological criteria. This study was carried out with the aim to evaluate if macroscopic features and cytologic formula can be used as potential diagnostic tool for distinguishing between malignant cells from reactive mesothelial cells in peritoneal effusions. We have examined the peritoneal effusions collected from 81 available cases, with a histological diagnosis known, from routine morphologic features. The various macroscopic parameters that were registered by macroscopic analysis included the registration of color, transparency and fluidity of peritoneal effusions. Comparing the results, there wasn`t found any relationship between peritoneal fluid containing cancer cells and liquid color. Cell smear appearance had a various cells populations and the quantitative analysis of effusions was not enough useful in establishing the final diagnosis. Keywords: peritoneal effusions, macroscopy, cytology, malign, benign __________________________________________________________________________________________ 1. Introduction The cytological diagnoses of serous effusions are usually made by routine cytomorphology with certainty, allowing treatment decisions. Various studies have shown a sensitivity of 57.3% and specificity of 89% by conventional cytology for the detection of malignant cells in effusion samples [1]. The conventional cytology rate for identification of neoplastic cells in effusions is about 60%. The rate of diagnostically equivocal effusions in routine cytology is dependent on the volume of effusion examined, type of preparation and staining, experience of the examiner, and application of ancillary methods [2]. Peritoneal effusions are a frequently encountered clinical manifestation of metastatic disease, with breast, ovarian, and lung carcinomas and malignant mesothelioma leading the list [3, 4]. Neoplastic cells that disseminate into cavities containing effusions are highly metastatic and possess ISSN-1453-1267 a strong autonomous proliferative drive while concurrently being stimulatory of exudative effusions. The diagnosis of a malignant effusion signifies disease progression and is associated with a worse prognosis regardless of the tumor site of origin. Furthermore, cancer cells of different origins differ considerably in their biology and have unique phenotypic and genotypic characteristics [5]. Primary cytomorphologic criteria of malignancy include cellular aggregates, pleomorphism (variable cellular appearance), anisocytosis (variation in cell size), anisokaryosis (variation in nuclear size), multinucleation, prominent to irregular nucleoli, increased nuclear to cytoplasmic ratio, monomorphic cellular appearance, and increased mitotic figures. Hyperplastic mesothelial cells also may exhibit anisocytosis, anisokaryosis, increased nuclear to cytoplasmic ratio, binucleate and multinucleate, and scattered mitoses. Any situation that results in fluid accumulation within the body cavities can induce mesothelial cell hyperplasia and exfoliation with an © 2010 Ovidius University Press The clinical utility of aditional methodes... / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010) abnormal cellular morphology [2]. Therefore, the differentiation between mesothelial cell hyperplasia and mesothelioma may be difficult or impossible. The first report of an intraoperative examination of peritoneal cytology to detect subclinical metastases was presented in 1971. Patients with normal peritoneal cytological specimens had better survival rates than patients with abnormal findings, but only one abnormal cytologic specimen was found in early stage disease. [6]. Factors such as in patient versus outpatient management and associated procedural discomfort are important in the decision making process, and the patient should participate in these subjective considerations [7]. In addition, the etiology of the primary complaint is frequently multifactorial. However, malignant effusions recur, and therefore repeated paracentesis, especially if the fluid rapidly reaccumulates, is usually not a good long-term solution unless the patient’s overall prognosis and current condition prohibits a more invasive option. It is difficult to compare results and determine the true efficacy of different techniques and agents because endpoints and response criteria as well as the extent and method of follow-up vary. Therefore various techniques should be used to increase the diagnostic accuracy of malignancy in serous effusions. centrifuging the peritoneal liquid samples at 1500 rpm for 5 minutes, using Shandon Cytospin preparations. After the centrifugation, the stained is fixed using alcohol (95% ethyl alcohol) as the fixative. Effusion cytology was studied from 40 peritoneal effusions associated with at least one malignancy and 41 effusions collected from pacients with hepatic cirrhosis. We have examined the peritoneal effusions from routine macroscopic and cytologic features. Determination (the qualitative method) of cellular density, specific weight and protein content from peritoneal fluid was performed by the Riwalta reactions [61]. Riwalta reaction is the reaction performed for differential diagnosis of exudates from transudates, based on precipitation of fibrin (insoluble protein, the main component of blood clot, a result of thrombin action on fibrinogen in plasma soluble, synthesized by the liver) meeting usual in inflammatory exudates (transudates usual, do not contain this fibrin) [10]. The reaction is positive when dripping the liquid examined in the mixture is obtaining an opalescent, as a cloud. For obtaining the liquid peritoneal cytology formula, 100 cellular elements were measured from each smear cellular, thus directly establishing a percentage value. 3. Results and Discussions From all 81 cases who developed peritoneal fluid, 41 were benign cases (associated with liver cirrhosis), 4 cases were associated with hepatic carcinoma, 4 cases with lung carcinoma, 18 cases with ovarian carcinoma, 4 cases with breast carcinoma, 9 cases with gastrointestinal carcinoma tract and 2 cases with peritoneal mesothelioma (Table 1). The studied cases were divided into two groups: the 41 benign cases were included in lot I and 40 cases of peritoneal fluid associated with cancer were included in lot II. 2. Material and Methods This study was based on evaluation of 81 available cases, with a histological diagnosis known, carried out in Emergency Clinical Hospital of Constanta – Pathological Anatomy Department (SCJUC ) from octobre 2007 to January 2010. Follow-up data were obtained from the Tumor Registry at SCJUC. Clinical charts of all the patients whose peritoneal fluid samples were sent for cytological examination during the study period were retrieved for relevant information. The fluid for cytological analysis was collected during laparotomy from the abdominal cavity. If no fluid was present, the peritoneal cavity was lavaged with saline solution, and the fluid was then collected for analysis. Giemsa stained and Papanicolaou stained slides were prepared from sediment obtained by Table 1. Peritoneal fluid distribution according to primary disease and the number of cases 158 Lots Primary disease Lot I (no=41) Hepatic cirrhosis No of cases 41 Ana Maria Creţu et al. / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010) Lot II (no=40) Hepatic cancer Ovarian cancer Gastrointestinal cancer Breast cancer Pulmonary carcinoma Peritoneal mesothelioma 4 18 9 4 3 2 In terms of etiology, the highest number of cases groupt in lot II were shown to have ovarian origin, represented by ovarian carcinoma (n = 18) (45%), followed by cases associated with gastrointestinal carcinoma (n = 9) (22.5%), liver and breast carcinoma (n = 4) (10%), carcinoma lung (n = 3) (7.5%) and malignant mesothelioma (n = 2) (5%) (Fig.1). The type of neoplasia associated with most cases with peritoneal effusions accumulation proved to be represented by ovarian carcinoma. Fig. 2. The peritoneal fluid on different intervals of age (HC hepatic cancer, OC ovarian cancer, GIC gastrointestinal cancer, BC breast cancer, PC lung cancer, MM malignant mesothelioma) According to cancer staging [8], - stage I: generally include small tumors without invasion and who are perfectly curable in most cases the prognosis favorable - stage II and III includes tumors with local invasion of surrounding tissues and lymph nodes, therapeutic approach and prognosis are different depending on the time cells and organ of origin, - stage IV: at this stage are in general inoperable tumors, metastasis and recurrence and a reserved prognostic survival, only 7 cases (17.5%) (2 / 2, and peritoneal mesothelioma 100% 5 / 18, 27.77% ovarian carcinoma) were rated as stage III, the rest (82.5%) fits into state IV, which shows that, as the stage progresses neoplasia, this is more prevalent peritoneal fluid (Fig.3). Fig. 1. Percentage distribution of cases according to the origin of cancer associated Most patients in Group II (32.83%) were within the range of ages 61-70 years (40%), followed by the 51-60 range (35%). Cases registered with ovarian carcinoma were included in most (61.11%) in the 5160 age range, those registered with gastrointestinal carcinoma and the liver were contained mainly in the 41-50 age range, breast cancers, malignant mesothelioma and lung were within the range 61-70. (Fig. 2). Fig. 3. Percentage of cases according to stage neoplasia 159 The clinical utility of aditional methodes... / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010) From all patients with histopathologic and clinical data that indicate malignancy, a number of 5 patients (2 / 2, 100% associated with malignant mesothelioma, 1/4, 25% associated with hepatic carcinoma, ¼, 25% associated with breast carcinoma, 1/3, 33.33% associated with lung carcinoma) were deceased before drawing to the final study (a period of approximately three years from the accumulation of fluid in the peritoneal cavity), in all cases, the peritoneal fluid cytology recorded the presence of malignant cells (Fig. 4). Thus, it was recorded: the extracted amount, the product color, transparency and its consistency. After macroscopic analysis, the most liquids from the group I was found to shown yellow color (from very light yellow to orange - yellow), and most fluid were transparent. In stead, the peritoneal effusions from group II had a variable macroscopic appearance: a number of 29/40 (72.5%) were intense yellow colored and transparents, many of them (17/29, 42.5% of all liquids associated with a carcinoma) had tissue fragments occupying approximately 25-50% from all quantity effusions, suspended in liquid; 5 (12.5%) showed a yelloworange fluid and opaque, and a total of six (15%) were hemorrhagic (deep red), fluid and opaque (Fig. 5). Of these, 19 / 40 (47.5%) had fragments of tissue suspended in peritoneal effusions. These fragments were then included in paraffin, stained and examined microscopically. We can say that the macroscopic analysis of peritoneal fluid, associated with cases of cancer are different from those associated with cases of cirrhosis only by this tissue fragments founded suspended in the effusions, with a capacity of discrimination of 47.5%. Fig. 4. Percentage of prognostic survival of patients included in the study (OC- ovarian cancer, GIC gastrointestinal cancer, HC - hepatic cancer, BC – breast cancer, PC - pulmonary cancer, MM malignant mesothelioma) Thus, the percentage of peritoneal fluid accumulation in the abdominal cavity is directly proportional to the tumor stage and also with the diagnosis of malignant peritoneal effusions, meaning that the progression of cancer is associated with an unfavorable prognosis. None of the patients with ovarian or gastric carcinoma were associated with an unfavorable prognosis, which indicates that this patients are available for a longer treatment. Neoplasia stage, histological grade of neoplasia, positive cytology of peritoneal fluid and patients age (61-70 years) were correlated statistically with the prognosis. The first step in the analysis of peritoneal fluid was represented by analysis of the macroscopic point of view of biological material (peritoneal effusions). Fig. 5. Peritoneal fluid: yellow (a) and hemorrhagic The registration of the important differences existing between macroscopic peritoneal fluids is essential, representing the first way in effusions discrimination (in transudates and exudates), so that, the material subjected can provide useful information for further evaluation of the cells from cytological smears. It is known that bleeding effusions (deep red) are often caused by a cancer and that these liquids often contain cancer cells [9]. 160 Ana Maria Creţu et al. / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010) However, comparing the results, after performing peritoneal fluid cytology, with those obtained by macroscopic evaluation, of the 40 effusions associated with at least one malignancy, only 6 (15%) were founded to be red colored (hemorrhagic), emphasizing that it does not exist any relationship between peritoneal fluid containing cancer cells and fluid color. After conducting the Riwalta reactions [10], the 81 peritoneal effusions were classified in: 29 (35.80%) transudates peritoneal fluid (with low cell density and low protein content, which is usually accumulated in benign conditions) and 43 (53.08%) exudate (effusions with high cell density and high protein content, which is accumulated most in malignant conditions), and 9 (11.11%) mixed, intermediate peritoneal effusions. Thus, peritoneal fluids were classified into three groups: group I (transudates), group II (intermediate, mixed) and group III (exudates) (Table 2). element in cytology grading of malignancy, and was quantified by mitosis counting. Fig 6. The benign or malignant nature of effusions after conducting the Riwalta reaction In smears classified as benign, isolated cells represented 90% of total cells, cell groups recovered to a rate of 10%. 5% were represented by free nucleus or cells with damaged cytoplasm. Mesothelial cells (33%) (33 cells of 100 elements) and lymphocytes (30%) were the majority cell type in the group of benign peritoneal effusions, followed by macrophages (17%), polymorphonuclear leukocytes (PMN) (9%) and erythrocytes (7%). Average of total number of mitosis founded in studied smears was 3mitosis/smears (Table 3). Cellular composition of effusions founded to be suspicious for malignancy was similar with the one of benign peritoneal effusions: mesothelial cells (28%) (28 cells of 100 items) and lymphocytes (26%) were the majority cell type, followed by neutrophils (13%), atypical cells, suspicious for malignancy (9%), erythrocytes (9%) and macrophages (7%). Average of total number of mitosis founded in studied smears was 7 mitosis/smears (Table 3). In the group of patients with malignant cancer, mesothelial cells represented 29% and erythrocytes 21%, followed by lymphocytes (18%), polymorphonuclear leukocytes (16%), macrophages (4%) and malignant cells (4%), average of total number of mitosis was 9 mitoses / cell smear (Figure 7). Table 2. Distribution of cases after Riwalta reaction Lots Lot I (n=41) Lot II (n=40) Primary cancer CB Transudates (N=29) 21 Mixed (N=9) 3 Exudates (N=43) 17 CH (4) 1 1 2 CO (18) CGI (9) CM (4) CP (3) MP (2) 3 1 2 1 0 0 2 2 1 0 15 6 0 1 2 Since only 26/40, 65% of peritoneal effusions associated with different type of cancer resulted to have characters of exudates, and only 21/41, 51.21% of effusions associated with liver cirrhosis were shown to be transudates, it follows that, by conducting the Riwalta reaction, it can determine the benign or malignant nature of effusions in a proportion of 58.10% (Fig.6). Cell smear appearance had a various cells populations and the quantitative analysis of effusions was not enough useful in establishing the final diagnosis. There were observed 7 cell types present in variable number. Proliferative capacity of tumor cells - tumor aggressiveness - is an important 161 The clinical utility of aditional methodes... / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010) Tabel 3. The percentage of cellular elements (%) Mesothelial cells Atipical cells Malignant cells erythrocytes, lymphocytes PMN macrophages mitosis / cell smear Average standard Deviation p(t<0,05)/BE* p(t<0,05)/AE* t(p<0,05)/BE* t(p<0,05)/AE* BE* 33 AE* 28 ME* 29 0 0 7 30 9 17 2 9 0 9 26 13 7 7 0 3 21 18 16 4 9 12,25 13,15566 12,375 9,738546 12,5 10,12776 0,983097 0,966668 0,98028 0,365912 0,359605 0,35831 determined by quantitative analysis, was variable regarding cell populations, and we could not get enough useful informations, so that the cytologic formula of peritoneal fluid shows no importance for the final diagnosis. 5. References [1]. BEDROSSIAN C.W.M., 1994 - Malignant effusions: A multimodal approach to cytologic diagnosis., Vol. 3:54-188, New York: IgakuShoin. [2]. MOTHERBY H., Nadjari B., Friegel P., et al., 1999 - Diagnostic accuracy of effusion cytology. Diagn. Cytopathol., 20:350-351. [3]. REDMAN C.W., Chapman S.E., Chan S.Y., et al., 1991 - Out-patient peritoneal lavage cytology in the detection of residual epithelial ovarian cancer. Cytopathol, 2: 291–298. [4]. JOHNSON W.D., 1966 - The cytological diagnosis of cancer in serous effusion. Acta cytological, 10:161-172. [5]. RUNYON B., 1999 - Approach to the patient with ascites. In: Yamada T, Alpers DH, Laine L, Owyang C, Powell DW, eds. Textbook of Gastroenterology. 3rd ed. Philadelphia: lippincott Williams & Wilkins, 966-991pp. [6]. CREASMAN W.T. & Rutlegge F., 1971 - The prognostic value of peritoneal cytology in gynecologic malignant disease. Am J Obstet Gynecol, 110: 773–781. [7]. THUNNISSEN F.B., Peterse J.L., Van Pel R., et al. 1993 - Reliability of fine needle aspiration cytology for distinguishing between carcinoma, lymphoma and sarcoma: the influence of clinical information. Cytopathology, 4:107. [8]. PETTERSON F., 1995 - International Federation of Gynecology and Obstetrics: annual report of the results of treatmant in gynecological cancer. Stocholm: Panorama Press AB, 83-227. [9]. SIMOJOKI M., 2003- Type I and type III collagen metabolites and peritoneal cells in predicting the clinical outcome of epithelial ovarian cancer patients, Oulu University Library, 136-179. [10]. POPA G., 1971- The cytodiagnostic by puncture in medical practice, Medical Press. *BE – benign effusions, AE – atipical effusions, ME – malignant effusions Fig.7. Effusions cells populations: 1 - benign, 2 atipical and 3 - malignant. 4. Conclusions Malignant peritoneal effusions contribute to considerable morbidity in cancer patients and generally portend an overall poor prognosis. Treatment of malignant peritoneal effusions is palliative; therefore, quality of life issues, as well as the risks and benefits of the therapeutic options, become more critical. Comparing results, after the peritoneal fluid cytology with those obtained by macroscopic evaluation was obviously that there was no relationship between peritoneal fluid containing cancer cells and liquid color. Cell smear appearance, 162 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 SPATIO-TEMPORAL DYNAMICS OF PHYTOPLANKTON COMPOSITION AND ABUNDANCE FROM THE ROMANIAN BLACK SEA COAST Laura BOICENCO National Institute for Marine Research and Development „Grigore Antipa” 300, Mamaia Bd., Constanta, 900581, Romania, e-mail: boicenco@alpha.rmri.ro __________________________________________________________________________________________ Abstract: Based on more than 2,000 samples collected during 1996-2007, the paper deals with the taxonomic and ecological composition, spatio-temporal development of phytoplankton blooms from waters of up to 50 m depths laying on the Romanian Black Sea. The author identified 396 species, varieties and forms, and assessed a density mean varying among 417 and 3,376∙103 cells∙l-1. Bacillariophyta phylum, with a number of 157 taxa and a density mean of minimum 186.4 (in 2000) and maximum 2,311.9∙103 cells∙l-1 (in 1997), was the most numerous (39.6% of the total); Dinoflagellata was the second dominant group, represented in the communities with 85 taxa (21.5%); density means ranged from 9.2 (in 2003) and 225.6∙103 cells∙l-1 (in 1997). Groups Chlorophyta and Cyanobacteria represented only 19.4 and 12.9%, respectively, from the total number of species. Species showing huge developments in the reference period were: the diatoms Skeletonema costatum, Cerataulina pelagica, Nitzschia delicatissima, Chaetoceros socialis, Cyclotella caspia and dinoflagellates Prorocentrum minimum, Heterocapsa triquetra and Scrippsiella trochoidea. Keywords: taxonomic composition, ecological composition, phytoplankton blooms __________________________________________________________________________________________ 1. Introduction The early 1990s seemed to be a new beginning for the Black Sea ecosystem. After the “Mnemiopsis era” describing the 8th decade, superimposed on the 20 years-long “eutrophication era” started in the 1970s, signs of improvement of its ecological state occurred, evidenced by a reduction of the Danube river nutrient input, a decrease in the frequency of hypoxia conditions, an increase in fodder zooplankton biomass, and a drop in M. leidyi’s abundance. The recovery of the ecosystem was attributed partly to the collapsing economy and agricultural production, and to some protective measures taken to control anthropogenic pollution in all the coastal countries. Due to their short life cycles and quick response to changes in their environment, the phytoplankton was sensitive to these new shifts, displaying a tendency to “normal” status before eutrophication: decreased amplitude and frequency of blooms, and a qualitative and quantitative structure similar to the period 1960-1970 rather than 1980-1990. ISSN-1453-1267 So, between 1991 and 1996, only six maximum densities, higher than 106 cells·l-1, were registered, compared to 13 in the1980s; among them, only two species produced ample bloom events: Prorocentrum minimum (53.1 and 93.7·106 cells·l-1 in the summer of 1991 and 1995) and Microcystis pulverea (60.0·106 cells·l-1 in the spring of 1991) [1]. The range of algal groups was different from that of 1970s and 1980s, but quite similar to that of 19601970 with a reduction of non-diatom bloom amplitude and increase of numerical density and especially biomass of diatoms. The reduction of non-diatoms coincided with decrease of nutrient stocks, especially of phosphates, which reached concentrations of 2.55 µM·l-1 in 1991-1996, 2.8 times lower than in 19861990 [1]. During 1995-1996, the Black Sea ecosystem showed abrupt shifts in all trophic levels, from primary producers to apex predators. This arose as a manifestation of concurrent changes in its physical climate induced by intensive warming of surface waters, as well as abrupt increases in the mean sea level and annual mean fresh water flux [2]. © 2010 Ovidius University Press Spatio-temporal dynamics of phytoplankton composition.../ Ovidius University Annals, Biology-Ecology Series 14: 163-169 The aim of the present paper is to evaluate the spatio-temporal dynamics of the main taxonomic groups, and also the main bloomforming species during 1996-2007. 2. Material and Methods Biological material was collected during seasonal surveys carried out within the scientific NIMRD’s programs, on board RV/STEAUA DE MARE, in the Romanian coastal waters laying between 43050’-45005’N and 28050’–30000’E (Fig. 1). 2,018 quantitative samples were collected from 472 stations covering the whole Romanian littoral at standard depths (0, 10, 20, 30, 40, 50 and 60m), from the following profiles: Sulina, Mila 9, Sf. Gheorghe, Zaton, Portiţa, Chituc, Constanta, Mangalia. microscopic processing, the sample is again siphoned off up to 10ml and stirring. 0.1ml of sample is examined under a ZEISS inverted microscope; the cells are counted and identified at species or genus and the numerical density is obtained relating the number of cells to a volume of 1 litre. Table 1 presents the environmental background (inorganic nutrients) of the annual phytoplankton developments. Phosphates showed a sharp decrease after 1997 down to a level similar to that before eutrophication. The inorganic total nitrogen concentrations have steadily depleted ever since the 1980s down to a minimum 9.48µM in 1985 followed by a relative long period, when these nutrients presented non-uniform oscillations. Among 1995 and 2005, the levels of total inorganic nitrogen homogenously decreased, the maximum limit being situated between 10 and 15µM. In recent years their concentrations have began to increase to more than 20µM, a value similar to that from 1980. With the exception of Si/N ratio, the molar ratios are still far by from the normal values, indicating that trophic anions do still not have optimal values for the normal development of marine phytoplankton, although they show a decreasing tendency. During the last period, the N/P ratio increased, due to an excessive decrease of phosphates and slight increase of inorganic nitrogen [5]. Table 1. Multiannual mean of surface nutrient concentrations in coastal waters off Constanta Period N-NO 3 (µM) N-NH 4 (µM) P-PO 4 (µM) SiO 4 (µM) Fig.1. Sampling network The sampling used a NISKIN bottle; the water is transferred in 500ml bottles and preserved with formaldehyde 4%. In laboratory, the samples were processed using the MOROZOVA-VODIANITSKAIA and BODEANU’s methods [3, 4]. After two weeks of sedimentation, the supernatant liquid is siphoned off up to about 100ml. The sample is put in a small jar for another 10 days sedimentation. Before 1983-‘90 6.90 5.11 6.54 11.0 1991-‘00 5.90 7.06 1.86 12.6 2001-‘05 7.98 6.12 0.49 13.7 3. Results and Discussions During 1996-2007, 396 microalgae species, varieties and forms belonging to seven phyla were identified in the Romanian Black Sea waters (Fig. 1), the minimum number of 140 being found in 1996 and the maximum one in 2004. The most important group is Bacillariophyta, with 157 species, representing 39.6% of the total; the second place is occupied by dinoflagellates, with 85 species (21.5%), followed by 164 Laura Boicenco / Ovidius University Annals, Biology-Ecology Series 14: 163-169 chlorophytes – 77 species (19.4%) and cyanobacteries – 55 species (12.9%); the rest of phyla (Chrysophyta, Euglenophyta, Cryptophyta), with 12, 8 and 6 species, respectively, constitute together only 6.6% of the total (Fig. 2). Fig. 2. Taxonomic composition. in the same year. The diatoms produced their highest mean densities during summer; in the summer of 1997, they exceeded 6 million cells per litre. When we were able to collect samples in winter, e.g. in 1999, we found out that many diatoms – such as S. costatum, C. caspia, Ch. socialis, N. tenuirostris began to vegetate in winter. So, we detected communities very well constituted, higher than 106 cells∙l-1. In five out of 12 springs investigated (1999, 2003, 2004, 2006 and 2007), the diatom populations were most abundant; they progressively decreased toward summer and autumn. Table 3 shows the diatom species with the highest densities in the Romanian Black Sea waters, between 1991 and 2007. Ecologically, the phytoplankton represents a combination of autochthonous species, comprising euryhaline marine and brackish water forms (218), and alochthonous forms, comprising fresh-brackish and fresh water forms (178), reflecting the mixed marine water masses and riverine fresh waters which characterise the hydrologic regime of the Romanian sector. Table 3. The highest densities of diatoms (106 cells∙l-1) 19962001Species 2000 2007 Cyclotella caspia Skeletonema costatum Nitzschia tenuirostris Cerataulina pelagica Chaetoceros socialis Skeletonema subsalsum N. delicatissima Table 2. Structure by ecologic groups. Phylum Bacillariophyta Dinoflagellata Chlorophyta Cyanobacteria Chrysophyta Euglenophyta Cryptophyta Total Marinebrackish 112 83 0 9 8 2 4 218 Freshbrackish 45 2 77 42 4 6 2 178 Diatoms During 1996-2007 the averaged data for the whole Romanian littoral waters suggest that the communities are dominated by diatoms, both in terms of numeric density and biomass. The multiannual mean of 833.5·103 cells·l-1 is 12.2 times higher than dinoflagellates (68.5·103 cells·l-1). The highest diatoms mean density 2,311.9·103 cells·l-1, achieved in 1997 was 10.2 times higher than that recorded for dinoflagellates 10.5 24.4 1.8 8.2 22.2 4.4 0.6 78.6 37.3 15.5 10.0 7.5 3.9 2.5 Small-sized diatom, Skeletonema costatum is an omnipresent species in the communities identified not only at the Romanian littoral but in whole Pontic basin, producing most of the bloom events. Its maximum level was registered in July 2002, off Constanta (37.3∙106 cells∙l-1). Its second outburst occurred in the shallow waters of Mamaia Bay (where the sampling is carried out almost weekly); starting in the second half of March 1998, the phytoplankton communities were more and more abundant, attaining the value of 24.3∙106 cells·l-1 on March, 31. We have to remark that the communities from Mamaia Bay were almost monospecific, being constituted up to 99.8% only by Skeletonema. Eurythermal and euryhaline species, Skeletonema vegetates abundant starting from winter, not only in Mamaia Bay, where usually attain over 5∙106 cells∙l-1, in January-March, but also in deeper waters off Constanta, where its concentrations reached 165 Spatio-temporal dynamics of phytoplankton composition.../ Ovidius University Annals, Biology-Ecology Series 14: 163-169 3.6∙106 cells∙l-1 in January 1999. In the spring of 2006, again in Mamaia Bay, S. costatum produced two other bloom events: 15∙106 cells∙l-1 (April, 25) and 11∙106 cells∙l-1 (May, 4) (Fig. 3), when the temperatures oscillated from 9.8 to 13.30C and the salinity decreased gradually from 16.53 to 12.64 and 9.04 PSU. In waters under the direct influence of the Danube, Skeletonema often produced densities ranged from 1.4 to 6.1∙106 cells·l-1, both in spring and summer. But in September 1999, its populations were even richer at all the stations of the profile: 6.0 (Sulina), 8.1 (Mila 9), 7.7 (Sf.Gheorghe) and 6.3 ∙106 cells·l-1 (Portita). Fig. 3. Long-term evolution of S. costatum blooms. However, the last blooms produced by Skeletonema are much lower than those recorded in the period of maximum eutrophication. A good indicator of hypereutrophic waters, S.costatum showed overwhelming populations after 1970; for instance, between 1983 and 1986, S. costatum bloomed up to its highest value of 141.4∙106cells∙l-1 in April 1983. But, the most significant bloom event registered during the whole study period was generated by another diatom, Cyclotella caspia in the shallow waters of Mamaia. On May 3, 2001, it reached the value of 78.6∙106 cells·l-1, which is 3.2 times higher than S. costatum’s peak; the event was amplified by the abundant population of Skeletonema, raising the total density up to 84.82∙106 cells∙l-1. Cyclotella gave another two important outbursts, but they were 7.6 and 4.0 times lower than the preceding one: in June 1999 (10.4∙106 cells∙l-1) at Mamaia, and June 2005 (19.7∙106 cells∙l-1) at Constanta (Fig. 4). Rich populations were found also in the northern sector, but never as high as those found in the southern area; the richest one was identified in waters from Sf.Gheorghe site (6.4∙106 cells·l-1). Anyway, these highest values are far from the exceptional development recorded by Cyclotella in 1981 (300∙106 cells∙l-1) [6]. Fig. 4. Long-term evolution of C. caspia blooms. Chaetoceros socialis is the third diatom with frequent occurrence and densities higher than 100∙103 cells·l-1, but only two blooms were higher than 10∙106 cells·l-1: in June 1997, in front of the Danube Delta (Mila 9) (15.9∙106 cells·l-1), and in May 2000, at Mamaia (22.2∙106 cells∙l-1) (Fig. 5). Ch. socialis is a new entry the list of bloomforming species. During the period 1971-1990, only Ch. similis f. solitarius developed large concentrations: 13.2∙106 cells∙l-1 (between 1970 and 1980) and 21.5∙106 cells∙l-1 in May 1988. Fig. 5. Long-term evolution of Ch. socialis blooms. However, in 1956, 1957 and 1961, SKOLKA cited Ch. socialis among the species producing some abundances higher than 106 cells·l-1 (its peak of 2.6∙106 cells∙l-1 was attained in June 1957); generally it accompanied other bloom-forming species such as S. costatum [7]. During the study period, another diatom group, including Cerataulina pelagica, Nitzschia delicatissima and N. tenuirostris, periodically contributed to increase the total phytoplankton abundances, and C. pelagica had a density range from 3 to 10 million cells per liter. But only two of 166 Laura Boicenco / Ovidius University Annals, Biology-Ecology Series 14: 163-169 Nitzschia’ species (N. tenuirostris and N. delicatissima) had occurred and had low densities. Apart from the N. tenuirostris’ single bloom produced in July 2006 in Mamaia Bay (15.5∙106 cells∙l-1), the two species had densities higher than 106 cells∙l-1 only in five and six years, respectively. N. tenuirostris started to vegetate intensely after 1981, and reached its amplest bloom in the summer of 1989 - 74.8·106 cells·l-1 [6]. eutrophication got stronger and stronger, up to a climax from 1981-1990, the species attained even more prodigious proliferations, up to the value of 807.6∙106 cells∙l-1 in July 1987. In fact, no other species would ever attain such densities as the Prorocentrum between 1971 and 1990. In the following years, the amplitude of Prorocentrum’s blooms decreased, but in July 1995 it reached a density of 93.7∙106 cells∙l-1 (Fig. 5), 8.6 times lower than its overwhelming density in July 1987 [1]. Dinoflagellates With a long-term mean of 68.5∙103cells∙l-1, the dinoflagellates comprised small percentages of the total phytoplankton, with a maximum of 17% in 2007; the highest mean density was almost 225.6·103 cells·l-1 in 1997. However, during two springs (1998 and 2007) the populations of dinoflagellates were denser, with a density mean of 455.9·103 cells·l-1. Between 1996 and 2007 a few species had concentrations higher than 10 millions cells per liter (Table 4) in different areas and years. Table 4. The highest densities produced by dinoflagellates (106 cells∙l-1) Species Scrippsiella trochoidea Heterocapsa triquetra Gymnodinium cf. aureolum Prorocentrum minimum 19962000 0.3 13.6 10.5 20012007 25.3 16.0 10.7 9.0 Mass growth of the Prorocentrum minimum, causing the water to turn red, was recorded for the first time in the summer of 1974 along the Romanian littoral; the phenomenon was repeated in summers 1975 and 1976. Prorocentrum was the first dinoflagellate species reacting to the sudden decrease in salinity (monthly average reached 13 PSU, at Constanta) and huge increase in the concentrations of phosphates and nitrates (18 and 11 times respectively higher than the period 19591960). Presence of such extraordinary blooms had never been noticed before: 181.5 (1974), 78.7 in the (1975) and 111.6∙106 cells∙l-1 (1976), southern coastal waters, from Navodari to Mangalia [8]. During the following decades, when Fig. 5. Long-term evolution of P.minimum blooms. Beside the diatom Skeletonema, P.minimum is the second most common species in the whole Pontic basin giving some of the highest blooms, especially in the NW sector. In our study period, P. minimum continued to have massive developments, but much lower than the previous ones: in June 1999 –10.4∙106 cells∙l-1 and July 2001 – 8.93∙106 cells∙l-1, both of them in Mamaia Bay. Here, up to 2001, during warm months, the species’ populations frequently exceeded 1 million cells per litre; then, the densities were lower and lower, sometimes disappearing from samples. Three other dinoflagellates reached concentrations higher than 10∙106 cells∙l-1, namely Heterocapsa triquetra, Scrippsiella trochoidea and Gymnodinium cf. aureolum (Table 2). After developments, reaching a few or ten thousands cells per litre in the 1970s, H. triquetra and S. trochoidea came to the list of the bloom-forming species, the first with a value of 97.6 ∙106 cells∙l-1 in the period 19711980, and the second one with a value of 25.8 ∙106 cells∙l-1 in the period 1981-1990. After a period (19911996) of insignificant concentrations (highest value of 1.9 ∙106 cells∙l-1) [1], Heterocapsa again reached high concentrations: 13.6∙106 cells∙l-1, in May 1998 (at Mila 9) and 10.3∙106 cells∙l-1, in April 2000 (in Mamaia Bay) (Fig. 6). All along Romanian littoral, but especially in the northern sector, Heterocapsa produced substantial densities, ranging from 2.0 to 167 Spatio-temporal dynamics of phytoplankton composition.../ Ovidius University Annals, Biology-Ecology Series 14: 163-169 5.0∙106 cells∙l-1. S. trochoidea and Gymnodinium cf. aureolum had only one single large bloom 25.2 (August 2001) and 10.1∙106 cells∙l-1 (April 2007 in Mamaia Bay), respectively. species of cyanophytes took place under increased values of temperature, simultaneously with decreased values of salinity and concomitant with optimal concentrations of nutrients [8]. The euglenophyte Eutreptia lanowii had a constant frequency of occurrence throughout the analyzed period, with maximum developments in June 2007 of 7.4∙106 cells∙l-1, in Mamaia Bay, and 2.8∙106 cells∙l-1 July 2002 off Constanta. 4. Conclusions Fig. 6. Long-term evolution of H. triquetra blooms. Other Groups Representatives of other groups did not achieve significant densities, only sporadically did some species dominate the communities, and cyanobacteria were the most numerous comparing with chlorophytes and chrysophytes. The species Merismopedia, Microcystis, Gloeocapsa, Oscilatoria and Aphanizomenon were the commonest and most frequent cyanobacteries (Table 4). Table 4. The highest densities produced by other groups (106 cells∙l-1) Species CYANOBACTERIA Microcystis orae Microcystis pulverea M. aeruginosa Phormidium sp. CHRYSOPHYTA Emiliania huxleyi EUGLENOPHYTA Eutreptia lanowii 19962000 20012007 1.0 1.5 27 272.0 26.7 15.0 1.1 1.3 1.1 2.4 7.4 Three species of Microcystis genus (M. pulverea, M. aeruginosa and M. orae) produced maximum densities between 12.8 and 271.9∙106 cells∙l-1, especially during summer of 2001-2003. The intense development of these three small-sized Despite of the mitigation in the pressure exerted by anthropogenic eutrophication (i.e. depletion of the inorganic nutrient concentrations down pre 1970 values) and Mnemiopsis’grazing, the signs of the ecosystem rehabilitation identified at the phytoplankton level occurred after 1990, seem to be very fragile and labile. That means if the necessary conditions (sudden salinity reduction, sudden increase in water temperature, high concentrations of specific biogenic compounds) are fulfilled, many of phytoplankters can produce ample blooms. Some of the species producing frequent and overwhelming blooms in the previous decades, carried on generating significant blooms also in our study period (i.e. S. costatum, P. minimum, C. caspia etc). Other species have newly entered the list of bloomforming species, especially small-sized cyanophyte – M. pulverea (occurred during the period 1991-1996), M. orae, M. aeruginosa, Synecocystis sp., Gloeocapsa crepidinium, but also some large-sized diatoms Navicula sp., Amphora sp., Tabellaria sp. (after 1996); all of them are alochthonous fresh-brackish species, introduced into the sea mainly by the Danube River. M. orae gave a density of 272∙106 cells∙l-1 in the summer of 2000, the highest density occuring after 1990. Many times in the past, some of the bloom events, especially these of huge concentrations, were followed by fish and invertebrate mass mortalities. We used to consider that the species blooming at the Romanian littoral were dangerous only due to the negative impact produced as consequence of oxygen depletion, reaching the threshold for lethal limits for invertebrates and fish. Such case took place in 1999, after a relatively high (10.4∙106 cells∙l-1) but longlasting bloom (June-July-August) produced by 168 Laura Boicenco / Ovidius University Annals, Biology-Ecology Series 14: 163-169 Cyclotella caspia, in Mamaia Bay. Huge quantities of adult gobies, sole, plaice and turbot juveniles were washed up on the beaches or caught in lethargic condition from the sea by fishermen. As a matter of fact, HALLEGRAFF (1995) considers that species vegetating in densities over 5∙106 cells∙l-1 are harmful, since phytoplankton hyperproduction leads to regular violations of the ecosystem carrying capacity and severe economic losses to aquaculture, fisheries and tourism operations [9]. However, some of algal species widely distributed at the Romanian coastal waters, such as Chaetoceros socialis, C. curvisetus, Dichtyocha speculum, Ceratium fusus, can seriously damage fish gills, either mechanically or through production of hemolytic substances. Other ones, such as P. minimum, Dinophysis acuta, D. acuminata, D, sacculus, D. rotundata, M. aeruginosa are considered potentially toxic species, having the capacity to produce potent toxins, like DSP (diarrheic shellfish poisoning), that through the food chain could cause a variety of gastrointestinal illness to humans [9]. The relationship between anthropogenic activities and changes in phytoplankton composition and diversity is one of the main objectives proposed in Harmful Algal Blooms research. Long time series of phytoplankton community storage in the NIMRD data base should be reconsidered related to HAB increase. [3] [4] [5] [6] [7] [8] [9] 5. References [1] BODEANU N., RUTA G., 1998 – Development of the planktonic algae in the Romanian Black Sea sector in 1981- 1996. In Harmful Algae, B. Reguera, J.Blanco, L.Fernandez, T. Wyatt (ed.) Vigo, Spain, 1997: 188-191. [2] OGUZ T., DIPPNER J.W., KAYMAZ Z., 2006 – Climatic regulation of the Black Sea hydro-meteorological and ecological properties at interannual-to-decadal time scale. Journal of Marine Systems, 6: 235254. 169 BODEANU N., 1987/1988 - Structure and dynamics of unicellular algal flora in the Romanian littoral of the Black Sea. Cercetari Marine, 20–21: 19–250. MOROZOVA-VODIANITKAIA N.V., 1954 The Black Sea phytoplankton, Tr. Sevastopol. Biol., 8: 11-99 (in Russian). BSC, 2008. State of the Environment of the Black Sea (2001-2006/7). Edited by Temel Oguz. Publications of the Commission on the Protection of the Black Sea Against Pollution (BSC) 2008-3, Istanbul, Turkey: 23-49. SKOLKA H.V., 1967 – Consideraţii asupra variaţiilor calitative şi cantitative ale fitoplanctonului litoralului românesc al Mării Negre. Ecologie Marină, Vol. 2: 193-293. BODEANU N., ROBAN A., USURELU M., 1981 – Elemente privind structura, dinamica şi producţia fitoplanctonului de la litoralul românesc al Mării Negre în perioada 1972Producţia şi productivitatea 1977). ecosistemelor acvatice. N. Botnariuc ed., Ed. Acad. Rom., Bucureşti: 42-50. BODEANU N., ANDREI C., BOICENCO L., POPA L.,. SBURLEA A, 2004 – A new trend of the phytoplankton structure and dynamics in the Romanian marine waters. Cercetari Marine, 35: 77-86. VELIKOVA V., MONCHEVA S.,. PETROVA D, 1999 – Phytoplankton dynamics and red tides (1987-1997) in the Bulgarian Black Sea. Wat. Sci. Tech., Vol. 39, No. 8: 27-36. Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 ASPECTS REGARDING THE BIODIVERSITY OF THE AQUATIC AND SEMI AQUATIC HETEROPTERA IN THE LAKES SITUATED IN THE MIDDLE BASIN OF THE OLT RIVER Daniela Minodora ILIE “Lucian Blaga” University, School of Sciences, Departament of Ecology and Environmental Protection, 5-7 Dr. I. Raţiu Street, 550012, Sibiu, Romania __________________________________________________________________________________________ Abstract: The present work analyzes the bio diversity of the aquatic and semi aquatic heteroptera belonging to four habitats, respectively lakes situated within the middle basin of the Olt River. From the collected biological material, consisting of 724 samples there were identified 20 species of aquatic and semi aquatic heteroptera. We want to mention the presence of the species Paracorixa concinna in the lake in Cincşor, here being the single and only one appearance of this species till now in the basin of the Olt River. The different conditions of the researched habitats are to be seen in the structure of the communities of aquatic and semi aquatic heteroptera. The similitude among the established communities is a quite a reduced one. Keywords: aquatic and semi aquatic heteroptera fauna, communities analysis, the middle basin of the Olt __________________________________________________________________________________________ 1. Introduction The aquatic and semi aquatic heteroptera lives in a great variety of habitats, from temporary swamps to big lakes, from brooks to small and big rivers, from continental waters to the surface of the oceans. The aquatic and semi aquatic heteroptera are consumers of the 2nd degree (the food base consisting of both dead and alive prey). The present work proposes to evaluate the bio diversity of the communities of aquatic and semi aquatic heteroptera from the researched lakes that are situated in the following units of relief: Perşani Mountains, Făgăraş Mountanns and Hârtibaciu Plateau. The lakes are presented as follows: SO1: Bottomless Lake (Mateiaş) The lake is situated in Perşani Mountains, having the following coordinates: 450 59’ 08’’ N, 250 20’ 20’’ E, at an altitude of 522m. It is to be found on the left slope of the Olt River, being placed in the storages of the terrace allowing in this way the supply of the lake from the phreatic water. The lake, having a surface of approximately 870m2 is surrounded by willows. Phragmites communis and Typha latifolia covers about 5% from the banks area. The vegetation above and under the water is about 55-60% of the surface of the lake. ISSN-1453-1267 There are to be found Lemna minor, L. trisulca, Spyrogyra sp., and also Ceratophyllum demersum, Myriophyllum spicatum (a little). It is interesting to be mentioned the appearance in this station of the species Sagittaria latifolia and Potamogeton lucens that are seldom met in the middle basin of the Olt. SO2: Bâlea Lake The geographic coordinates are: 450 36’ 10’’ N, 0 24 36’ 49’’E, at an altitude of 2036m. It is a typical glacier lake sheltered in the so called Bâlea bucket, nearby the separating limit between the glacial circle and the former glacial valley. There is a mixed supply, this being the spring of the river Bâlea. The lake has a surface of 4.6 ha and a maximum depth of 11.35. S03: Cincşor The lake is situated in the Hârtibaciu Plateau, having the geographic coordinates as follows: 450 49’ 36’’ N, 240 49’55’’ E, at an altitude of 422m. It is an abandoned meander of the Olt River, which when there are big flood keeps in touch with the actual course of the river, being situated in its major riverbed. The supply of the lake is both from underground as well as superficial. © 2010 Ovidius University Press Aspects regarding the biodiversity... / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010) The banks of the river are covered with willows (Salix alba, S. triandra, S. fragilis), which make the banks more stable and also give shadow to the water. SO4: Netuş river Olt (Ilie, 2009). Here is the only station where appeared the species Ilyocoris cimicoides, its presence being linked to the under water vegetation. Other species of aquatic heteroptera (Sigara striata, Sigara iactans, Notonecta glauca, Plea minutissima) The Netuş Lake is situated in the Hârtibaciu Plateau having the coordinates as follows: 460 03’ 55’’ N, 240 47’ 55’’ E, at an altitude of 484m. The lake was arranged by people, its purpose being to reduce the flood. It also has a fish breeding interest, being placed in the major riverbed of the Hârtibaciu River. The vegetation is undeveloped. are also well represented from the same reason. On the other side, the vegetation above the water is favorable for the semi aquatic species (Microvelia reticulata, Mesovelia furcata and Mesovelia vittigera). The community of the aquatic and semi aquatic heteroptera from Mateiaş is defined by high values of the relative abundance of the species Ilyocoris cimicoides (A=30.31%), Microvelia reticulata (A=20.85%), Gerris argentatus (A=16.62%) and Sigara striata (A=12.54%) and values less than 10% for the other species. There is to be noticed an equilibrate structure of the heteroptera community as two species of aquatic heteroptera and respectively two species of semi aquatic heteroptera represents about 40% from the total of the community. On assembly the aquatic heteroptera represent 60% and the semi aquatic heteroptera about 40% from the heteroptera community in the lake in Mateiaş (in the terms of relative abundance). The species Notonecta glauca is represented by an average number of individuals, the dimensions of the population being determined by the big size and the predator behavior, which is extremely active. At the Bâlea Lake (SO2) we identified only two species of heteroptera although there was done the some number of gatherings, the habitat being of the same kind (natural lake) and the relief unit the same, namely mountain. This fact is a result of the great differences of altitude, which implies climate differences (especially the temperature, on which depends the existence and the development of the insects) as well as the vegetation (this being mainly a shelter against the predators). There was also noticed the fact that the species that were present in the Bâlea Lake are to be found in the Mateiaş Lake, too. At Cincşor (SO3) there were identified 10 species of aquatic and semi aquatic heteroptera. The most of the species belong to Corixidae family (4 species). The other families are represented by one or maximum two species. In the aquatic and semi aquatic heteroptera community of the Cincşor Lake, Micronecta scholtzi 2. Material and Methods The biologic material was gathered during September-October 2001, September 2002, August and September 2004, From three stations there were gathered two samples: in September and October 2001 from SO!, in September 2002 and August 2004 from SO2, in September and October 2001 from SO3. From the station SO4 there was done only one gathering (October 2004). For the identification of the species we used the determination key of the following authors: [1], [2], and [3]. There was calculated the relative abundance of each and every species from the researched habitats, diversity indexes ά - Margalef (for general aspects, such as the number of species and the number of individuals) and Lloyd-Ghelardi (for the evaluation of heterogeneity) – and the indicator of percentage similitude Renkonen, in accordance with [4]. 3. Results and Discussions As a result of the gatherings done during the periods mentioned before we identified a number of 20 species, from which 13 species are aquatic heteroptera (Heteroptera: Nepomorpha) and 7 species are semi aquatic heteroptera (Heteroptera: Gerromorpha), belonging to 9 families, presented in a number of 724 samples (table 1). The Corixidae family is the best represented taking into account the number of species (8 species), but considering the number of the gathered individuals the Naucoridae family is on the first place (202 samples). At Mateiaş (SO1) we identified 17 species representing 50% from the total number of species that were gathered in the middle basin of the 172 Daniela Minodora Ilie / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010) registered by far the highest value of the relative abundance (A=46.00%). Some authors considered important the fact that fish eat several species of heteroptera, especially Corixidae, reducing in this way their populations [5]. The populations of the species Micronecta scholtzi were noticed in the shore Table 3. The values of the Renkonen index S01- S01- S01- S02- S02- S03S02 S03 S04 S03 S04 S04 R. 23.87 19.40 21.00 0.00 22.22 0.00 The similitude between the aquatic and semi aquatic heteroptera communities in the 4 habitats was area of the aquatic habitat, a shadowed area and without underwater plants, having a reduced depth, which is not favorable for fish, this being the explanation of the abundance of this species in the heteroptera community. We also want to notice the presence of the species Paracorixa concinna, here being the only once it was registered till now in the basin of the Olt River [6]. At Netuş (SO4) there were identified two species of semi aquatic heteroptera: Gerris lacustris, being collected 7 individuals and Microvelia reticulata, 2 individuals being collected. In this case the number of species is a much reduced one because the ecologic conditions are not proper for these heteroptera. We want to notice that both species are semi aquatic ones, these being less sensitive than the aquatic species regarding the volume and the density of the underwater as well as the floating vegetation. established having as a base the Renkonen index, calculated with data of relative abundance of the species. It came out that there was a quite low similitude (table 3). 4. Conclusions There were identified 20 species, from which we noticed the species Paracorixa concinna, at Cincşor being the only registration in the basin of the Olt River. The number of the identified species in every habitat differs quite a lot (among 2-17 species) as well as the abundances of different species within the communities that establish them in those habitats (for example in SO1, the only station where the species Ilyocoris cimicoides was present, this being also the most abundant; in SO3 Micronecta sholtzi registered by far the highest value of the relative abundance). These show the variety of the conditions that are existent in those lakes; for the aquatic and semi aquatic heteroptera the quality of the habitats is connected with the altitude, damming, the development of the aquatic vegetation, the fish population, etc. The similitude between the communities of aquatic and semi aquatic heteroptera established in those 4 habitats is quite a low one. Table 2. The values of the diversity indexes ά obtained for every collecting station S01 S02 S03 S04 Index / Station Margalef Lloyd-Ghelardi 2.46 3 0.68 6 0.91 0 0.91 8 2.30 1 0.71 3 0.45 5 0.76 4 The values of Margalef index are quite high for the habitats SO1 (2.463) and SO3 (2.301) respectively low for the habitats SO2 (0.910) and SO4 (0.455) (table 2). The higher values of the index show that there were better conditions in the habitat for the heteroptera species. The Lloyd-Ghelardi index, varying between 0 and 1, shows for the researched habitats a relatively homogenous repartition of the individuals on the species, representing around 70% of the optimum value. SO2 is an exception having a higher value because of the identification of individuals number closed to the species number. 5. References [1] DAVIDEANU, ANA, 1999. Contribuţii la studiul heteropterelor acvatice din România, Teza de doctorat, Univ. ”Al. I. Cuza”, Iaşi, 427 pp. [2] JANSSON, A., 1986. The Corixidae (Heteroptera) of Europe and some adjacent regions, Acta Entom. Fennica, 47: 1-92. [3] POISSON, R., 1957. Hétéroptères aquatiques (Faune de France), 61: 1-263. [4] SÎRBU, I., BENEDEK ANA MARIA, 2004. Ecologie practică, Univ. Lucian Blaga, Sibiu, 1264. 173 Aspects regarding the biodiversity... / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010) [5] PAPÁČEK, M., 2001. Small aquatic and ripicolous bugs (Heteroptera: Nepomorpha) as predators and prey, Eur. J. Entomol., 98: 1-12. [6] ILIE, DANIELA MINODORA, 2009. Heteropterele acvatice şi semiacvatice din bazinul mijlociu al Oltului, Ed. Altip, Alba-Iulia, 1-279. 174 Daniela Minodora Ilie / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010) Table 1. The identified species of aquatic and semi aquatic heteroptera from the researched habitats and the values of the relative abundance Gathering Station Taxon Fam. Gerridae Gerris argentatus Gerris odontogaster Gerris lacustris Fam. Veliidae Microvelia reticulata Fam. Hydrometridae Hydrometra stagnorum Fam Mesoveliidae Mesovelia furcata Mesovelia vitigera Fam. Corixidae Micronecta (Dichaetonecta) scholtzi Corixa punctata Hesperocorixa linnaei Paracorixa concinna Sigara (Retrocorixa) limitata Sigara (Sigara) striata Sigara (Subsigara) iactans Sigara (Vermicorixa) lateralis Fam. Naucoridae Ilyocoris cimicoides Fam. Nepidae Nepa cinerea Fam. Notonectidae S01 Individuals number A% S02 Individuals number A% S03 Individuals number 110 16,62 3 1 0,45 0,15 138 20,85 1 0,15 11 22,00 21 10 3,17 1,51 4 1 8,00 2,00 23 46,00 6 0,91 2 0,30 1 2,00 1 2,00 1 2,00 1 2,00 1 0,15 83 12,54 45 6,80 202 30,51 2 0,30 5 A% 1 A% 10,00 33,33 174 S04 Individuals number 7 77,78 2 22,22 Aspects regarding the biodiversity... / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010) Notonecta viridis Notonecta glauca Fam. Pleidae Plea minutissima Individuals number per gathering station Species number per gathering station 2 20 0,30 3,02 15 2,27 2 66,67 2 4,00 662 3 50 9 17 2 10 2 175 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 PROGRAM OF PREVENTION AND CONTROL OF FUNGUS INFESTATION OF GRAIN AND FODDER , HUMAN AND ANIMAL PROTECTION AGAINST MYCOTOXINS drd.ing. Ioan Aurel POP*, conf. dr. Augustin CURTICĂPEAN**, drd.ing. Alin GULEA*, dr. Cornel PODAR*, ing. Iustina LOBONTIU*. * Staţiunea de Cercetare Dezvoltare penru Creşterea Bovinelor Mureş, str. Principală 1227, Sângeorgiu de Mureş, jud Mureş. ** Universitatea de Medicină şi Farmacie Târgu Mureş __________________________________________________________________________________________ Abstract: mycotoxins contained in forages may yield to cause different health issues on farm livestock as decreasing the forage intake and bioconversion, serious illness and death. Food and Agriculture Organization (FAO) appreciates on global level that 25% of agricultural products are contaminated with mycotoxins. These compounds contaminate feeds before and after harvesting. Food quality monitoring on each stage, especially due to it’s fungal potential risk is very important for the development of antifungal strategies adapted to local conditions. Thus, through a research project witch involves the quantification of mycotoxins concentrations from feed and food samples taken from different farms located in Central Region of Transylvania we managed to develop a new method of detection and quantification of three mycotoxins. The paper work presents a part of activities performed in a research project and comprises their results on preventing and control of funguses and mycotoxins. Keywords: mycotoxins, fungus, crops, methods. __________________________________________________________________________________________ 1. Introduction Food safety has become one of the directions very important area to protect and improve the quality of life. To ensure all elements contributing to the increase of consumer protection and food quality, develop new methods of control, as simple, low resource consuming, while used in normal conditions [1]. Thus, eliminating sources of toxic advanced occurring in food composition is a major goal. One such source is the species of fungi producing mycotoxins, which are found in most foods of plant origin whose storage / storage is inadequate, but worse is that we find and their metabolites in animal products, products from infested feeding. Monitoring primary storage conditions, and assessment on a representative sample of infestation by specific analysis will recommend specific methods of prevention / treatment of developing adverse effects of mycotoxins in crops. In a research project has developed a new ISSN-1453-1267 method for detection and quantification of three mycotoxins for monitoring the infection status of feed and food grain with mycotoxins in various units and areas located in Region Development Centru. [2] The paper also presents results of experiments: - Study the behavior of wheat, barley, triticales and corn hybrids tested in comparative culture from the years 2008, 2009 from SCDCB Mures and their hierarchy according to their resistance to disease attack; - Testing of eight plant protection products for disease prevention and control in cereals in climatic conditions in 2009 and monitoring the behavior of fungicides in the production. 2. Material and Methods Thurough the research project "Complex program of prevention and control of fungus infestation for grain and fodder for providing animal © 2010 Ovidius University Press Program of prevention and control of fungus… / Ovidius University Annals, Biology-Ecology Series 14: 177-180 (2010) wealth and consumer protection’ has achieved a status of monitoring infestation of feed and food grain with mycotoxins in various units and sectors Ardeal 1 Magistral Renan Exotic Gasparom Turda 14/98 Apullum located in the Development Region Center. Action was initiated in early June in a maximum period of susceptibility to the incidence of mycotoxins deposit being made by the team of researchers from SCDCB Mures Tg Mures and SC AGROFITOPLANT PharmacyLtd. Sampling activity was made taking account of Regulation (EC) NO. 401/2006 laying down the procedures for sampling and analysis methods for official control of mycotoxins in food. In total 180 samples were taken from 44 production units, which have in operation a total: 14 450 ha of arable land, 8960 cattle, 29,170 porcine, 36,640 birds, 8792 sheep. Average area of farms covered operating is 328 hectares. Of the total samples: 145 samples were concentrated (maize grain, flour, PVM's, milk powder) and 35 samples of forage (silage, half hay, hay, grains, beet chips, etc.). [1] The quantification of mycotoxins in the samples, at the UMF Mures (Mures University of Medicine and Pharmacy) has developed a new method to quantify the simultaneous separation of mycotoxins by liquid chromatography HPLC using a DIONEX Ultimate 3000 with UV detection simultaneously on different channels. Optimization method was performed to determine simultaneously, using an ordinary system, more relevant mycotoxins present in samples of feed corn stored for eight months. Based on production and behavior have established multi fenophasic comparative cultural components subject to this project, DC M01 and M02 with wheat varieties, triticale and barley. (Table 1) II II I GRAU Ariesan(Mt Producti a medie (Kg/ha) 6920 6780,9 7243,5 Isc 120,20 161,01 130,59 114,56 133,56 122,90 III VI II IV 6671,0 6851,8 105,98 110,33 VIII VII TRITICAL E Plai(Mt) Titan Trilstar Stil 00474T1-1 7699 7798,7 7971,4 7501,8 7590,3 7632,1 120,20 123,85 104,89 117,51 125,30 III II V IV I ORZ Gerlac(Mt) Regal Plaisant 6481 6570,8 6683,2 6189,1 110,00 97,12 98,81 I III II For testing resistance to major pests and diseases of maize hybrids grown in the area was established in late April (2008.2009) a crop of corn hybrids compared with 24 (S = 1000 m) in the experimental field of the resort located in Sg Mures. The main observations made: plant vigor, flowering time, date of silk, drought resistance, Helminthosporium sp., Puccini sp., Ustillago sp. Attack of Fusarium sp., The number of sterile plants, the number of broken and fallen plants, resistance to attack pest and grain production. The content determination of mycotoxins was performed at UMF Targu Mures (University of Medicine and Pharmacy. Mures) SPC Mures (Mures Public Health Center) Promovert laboratories in Champagne, France (company Bayer). [1] Table 1. Crop ranking regarding yield DON and ZON. Specia/Soiu l 7159,1 6536,0 7047,9 7070,5 Ierarh i zare 3. Results and Discussions Precision method for determining meets the minimum relative standard deviation (with values in a field of ± 15%) for quality control samples measured V I 178 Ioan Aurel Pop et al. / Ovidius University Annals, Biology-Ecology Series 14: 177-180 (2010) in both samples the same day and comparisons between samples from different days. Minimum limits of quantification for the three analytes / mycotoxins (2.88 ng / mL AflaB1, 2.88 ng variants examined only version control - untreated with fungicide containing deoxynivalenol was above the limit of quantitation of 220 ng / g, respectively 440 ng / g value in joining the legal permissible limit of 1250 ng / g DON. [1] / mL respectively OchrA 14.4 ng / mL Zeara) met the requirements of precision and accuracy so that the relative standard deviation to be included in a field ± 20% for both measurements on the same day as well as those performed on different days and the difference between mean calculated and nominal values (BIAS%) is also contained in a maximum field of ± 20%. Records on the evidence provided by corn and also on samples from various forage plants show an infestation of their importance to all three classes of mycotoxins, so Aflatoxin B1 and Ochratoxin A with zearalenone. Infestation levels are relatively high, regulated levels overruns are much more frequent and more significant if the first two Mycotoxins Aflatoxin B1 respectively Ochratoxin A. Thus, the calculated values for concentrations of mycotoxin present in almost all unknown samples analyzed exceed permissible concentrations and regulated at European level. [2] High levels of mycotoxins found in animal feed and is probably due to the chosen period when this work started in early June, during which cereal stocks are running, close grain with a one year old storage warehouses before the process is Cleaning for storing grain harvest. Using production data obtained, the observations on different varieties fenophase attack on disease resistance, deoxynivalenol and zearalenone content in samples taken at harvest 16 varieties of cereals were ranked using a synthetic index calculated Isc this. Results show that there are differences between varieties in terms of mycotoxins but not the values obtained exceeding the maximum allowed by law (1250 ng / g DON, and 100 ng / g Zon). Maize, based on production, moisture at harvest, percentage of plants broken and fallen and observations of vegetation during the attack on disease resistance of a synthetic index was calculated to ease the process generally ranking of cultivars. Results of tests carried out in laboratories SC Bayer SRL Promovert in Champagne, France reinforce the lessons learned so far. Of the seven 101 100 100 100 % spice sanatoase 100 99 98 97 97 97 Nativo 300 SC Falcon 460 EC 97 96 95 95 94 93 92 Martor netratat Folicur Solo 250 EW Tilt 250 EW Duett Ultra Prosaro 250 EC Produsul Fig. 1. Fungicides effect in Fusarium removal from Ariesat wheat variety at Tg Mures. 4. Conclusions Interpreted data show that the current methodology for preparing samples for analysis / quantification of mycotoxin content of substances of category has limits too generous. Thus, extraction of these substances (of which there are complex matrices) respecting the standardized methods, shows a lower sensitivity, which leads to highlighting of quantities / concentrations lower than actual. .[2] The existence of evidence over the maximum levels allowable by law certify the importance of this research and the need for a regional research antimycotic. Climatic conditions of the agricultural year 2008 - 2009, characterized by high temperatures throughout the crop growing season and low rainfall than -114 mm limited attack foliar and ear diseases, and the effect of crop protection products was not very visible. For further research would require more years of study to catch different climates. Large assortment of hybrid corn study allows farmers to select hybrids with high production potential and adaptability to the conditions of the area. To limit the attack of diseases and in particular Fusarium in seed must be transmitted primarily by limiting attack Pyrausta which facilitates infection 179 Program of prevention and control of fungus… / Ovidius University Annals, Biology-Ecology Series 14: 177-180 (2010) how damaging fungal diseases of plants and causes breaking of preventing deployment of mechanized harvesting in good condition. [3] 5. References [1] POP I., GULEA A., CURTICĂPEAN A., PODAR C. , 2009 - Program complex de prevenire şi combatere a infestării cu miceţi la cereale şi plante furajere pentru asigurarea bunăstǎrii animalelor şi protecţia consumatorilor, Raport de progres Transa a II-a. [2] A. CURTICĂPEAN, FELICIA TOMA, MONICA TARCEA, MANUELA CURTICĂPEAN, VICTOR SĂMĂRGHITAN, I. POP, A. GULEA, 2009 - Optimizarea unei metode HPLC de separare şi determinarea simultană a unor micotoxine din porumb,- Noi tendinţe şi strategii in chimia materialelor avansate. Institutul de Chimie Timişoara, Timişoara. [3] Pop I., Gulea A., Curticăpean A., Podar Cornel, 2009 - ‚Program complex de prevenire şi combatere a infestării cu miceţi la cereale şi plante furajere pentru asigurarea bunăstǎrii animalelor şi protecţia consumatorilor’- Raport de progres Transa I. 180 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 DATA ON THE DYNAMICS OF SOME MICROBIAL GROUPS IN SOILS WITH DIFFERENT TROPHIC STATUS IN CUMPĂNA REGION (DOBROUDJA) Elena DELCĂ Ovidius University of Constanţa, Faculty of Natural Sciences and Agricultural Sciences Mamaia Avenue, No. 124, Constanţa, 900527, Romania, Doctoral school Biology, Specialization Ecology __________________________________________________________________________________________ Abstract: The aim of paper was to assess the effect of administration of organic amendments on the dynamics of the abundance of microbial groups significant in nutrient cycling in soils. Abundance of total culturable bacteria ranged from 19.93x106UFC/g dry soil, to 501.79x106UFC/g dry soil. When soil was supplemented with manure microbial density showed a significant increase 501.79x106UFC/g dry soil compared with control variant. Bacterial density increased significantly as value, too, following the administration of specific biofertilizers (Biovin, Bactofil Professional; Mycos Green), up to 142.13 x106UFC/g dry soil. Inorganic fertilizers did not have a positive effect on microbial density values, being more or less similar to those reported for the control. Our preliminary data show that organic amendments with complex composition have a direct effect on the abundance and diversity of soil and influence indirectly the microbial metabolism and nutrient cycling rate. Keywords: humus, microorganisms, bioactivators, fertility _________________________________________________________________________________________ 1. Introduction To start and propose a suitable biological soil reconstruction plan it was necessary to initiate a series of observations and experiments in a characteristic agroecosystem of Dobroudja (Cumpana commune) in order to assess the current biological status. Using new agricultural technology, and adding different fertilizers the experiments have the aim to improve the number and activity of soil microorganism and indirectly to enhance the rate of organic matter decomposition. This would improve over time the soil structure and restore the stock of humus in the soil. 2. Material and Methods The experiments have taken place on a 7.5 ha plot situated in the outside of Cumpana, in Constanta district. Josef wheat was cultivated on the entire area, which was divided in 7 variants, each variant being administered a different type of fertilizer in different quantities and periods, as follows: ISSN-1453-1267 - Variant I - only chemical fertilizers - 100kg/ha N 15 P 25 K 15 in autumn, 150kg/ha NH 4 NO 3 at the beginning of spring; - Variant II – Biovin organic fertilizer 400kg/ha and Biovin 30 of l/ha, ½ at herbicide stage and ½ at flour stage; - Variant III – garden soil - 15t/ha in autumn; - Variant VI – l/ha of Biovin 30, ½ at herbicide stage and ½ at flour stage; - Variant V – Biovin 150kg/ha administered during sowing, 150kg/ha NH 4 NO 3 , 40kg/ha at the beginning of spring, 50kg/ha at herbicide stage and 60kg/ha at flour stage; - Variant VI – Biovin 375kg/ha, liquid Biovin 30 of l/ha, ½ at herbicide stage and ½ at flour stage, 1mc Green Mycos, 1l Bactofil Professional; - Variant – March – were not applied amendments. Biovin Fertilizers are being administered for the first time in Dobrogea. Biovin is being produced through a technological process from grape kernels. 12 years of western research proved the following: it aerates the soil, improves it (it contains up to 70% humus makers), and purveys all plants with nutritive elements and biostimulators, it enriches the soil with © 2010 Ovidius University Press Data on the dinamics of some microbial groups... / Ovidius University Annals, Biology-Ecology Series 14: 181-184 (2010) microorganisms that create humus, it strengthens the roots and it multiplies the percentage of smooth roots and radicular wintergr; Bactofil Professional is a product for improving the soil biological quality and contains nitrogen fixing bacteria phosphate-solubilization bacteria, and heterotrophic bacteria that stimulates the decomposition of organic matter. Green Mycos is a product containing arbuscular mycorrhizal fungi and a number of factors that stimulate the establishment of symbiosis, improving the soil quality up to 20 years. [1] Experiments began in autumn 2009 by sampling the soil at a depth of 15 cm approximately, followed by quantification of some agrochemical (humus, indice N 2 ) and biological parameters (bacteria, free N 2 -fixing bacateria, actinomycetes, microfungi). Quantitative determination of microbial abundance was done by decimal dilutions of soil followed by inoculation of known quantities on solid nutrient media. For this purpose, after weighing the samples were inoculated on culture medium with a specific composition. Thus, to determine the number of total culturable heterotrophic bacteria it has been used nutrient: - agar medium [2], [3] [4] - (pulvis yeast extract 2.5 g, peptone 0.2 g, Agar 17-20g. It was sterilized 20 min at 120oC); - free N 2 -fixing bacteria on Ashby medium, [5] (15g Mannitol; g K 2 HPO 4 0.2, MgSO 4 ∙ 7H 2 O 0.5 g, 0.2 g NaCl, CaSO 4 ∙ 7H 2 O 0.1 g, CaCO 3 5g, Agar 17-20g. was sterilized 30 min at 115oC). Determination was made on the environment actinomicete Czapeck – Dox ( 3g NaNO 3 ; 1g K 2 HPO 4 ; 0,5g MgSO 4 ; 0,5g KCl; FeSO 4 traces; Sucrose 30g; 17-20g Agar; pH 5,5; it was sterilized 30 min at 115oC) [3], [4], [7] and the abundance of microfungi was determined on Sabouraud medium (CaCl 2 0.5g, 0.1g K 2 HPO 4 , KH 2 PO 4 0.1g, 10% MoO 3 0.1ml, 0.05ml FeCl 3 10%, was sterilized 30 min at 115oC). The total number of bacteria per gram of soil was calculated using the formula: no. bacteria, actinomycetes, microfungi = X colonies x dilution x 10 x 100/100-U where X = average of colonies grown on culture medium, 10 = balancing coefficient of 0.1 ml of inoculum in the reporting of dilution soil U% = soil moisture. [8] 3. Results and Discussions The initial estimations have revealed a relatively low abundance variability between different experimental variants. Thus, the lowest abundance was detected in variant VI, heterotrophic bacteria having a mean abundance of 19.93 x 106 CFU/g dry soil (Fig. 1). The abundance was highest instead on variant II, in which case the total number of heterotrophic bacteria reached 45.45 x 106 CFU / g dry soil (Fig. 1). Fig. 1 Bacterial density distribution in the initial stage of the experiment (October 2009) Changes in microbial abundance in experimental and control reflect the heterogeneity of normal physicochemical and trophic conditions of the soil, the values recorded can be considered normal for chernozem soil type. Fig. 2 Distribution of heterotrophic bacterial density after six months of application of amendments (May 2010) 182 Elena Delca / Ovidius University Annals, Biology-Ecology Series 14: 181-184 (2010) After six months of application of organic and inorganic amendments microbial abundance showed considerable changes in some cases, so the highest density was recorded in the experimental group fertilized with manure, variant III, in which case we determined a density of 501.79 x 106 CFU/g dry soil (Fig. 2). The number of bacteria also increased significantly in variant VI up to 142.13 x 106 CFU/g dry soil (Fig. 2). Paradoxically, after six months I have noticed decrease of heterotrophic bacteria in variant I, which might be explained by the effect of administration of chemical fertilizers and organic substance consumption by bacteria. In autumn 2009 the initial amount of organic matter in the form of crop residue remaining after harvest decreased gradually as the decomposition and microbial consumption progressed and provide sufficient nutrients to maintain viable bacterial population as numerous as in the beginning of the experiment. In other variants (II, IV and V) microbial density presented weakly peaks compared with the control, and ranged between 30.73 x 106 CFU/g soil dry and 46.31 x 106 CFU/g soil dry (Fig. 2), situation observed also in control variant. At the beginning of the experiment, density of free nitrogen-fixing bacteria was relatively low, (Fig. 3), ranging from 10.3 x 105 CFU/g dry soil to 26.7 x 105 CFU/g dry soil. In case of variants I, III, V and VI values are very close to those recorded for control (Fig. 3). The highest number recorded for variants II and IV ranging between 22.7 x 105 CFU/g dry soil and 26.7 x 105 CFU/g dry soil, rely on local trophic conditions. In any case, there was a certain uniformity of abundance of nitrogen-fixing bacteria beginning of the experiment. At this stage relatively low abundance of nitrogen-fixing bacteria could be due to higher quantity of organic substance that stimulates competition within heterotrophic bacterial populations. Fig. 4 Change in binding density micro N 2 , after six months (May 2010) After six months of adding the fertilizers our estimations revealed a significant increase of the number of nitrogen-fixing bacteria, including that of the control (Fig. 4). Most evident increase of abundance of this group occurred I in variant III, where we recorded about 195.81 x 105 CFU/g dry soil (Fig. 4). Significant increases were recorded in the case of variants II, IV and VI, where abundance ranged from 90.44 x 105 CFU/g dry soil to 127.39 x 105 CFU/g dry soil (Fig. 4). In variant I and V, values were close to the abundance determined for control (Fig. 4). Table 1. Agrochemical analysis conducted in autumn 2009 Fig. 3 Changes of abundance of nitrogen-fixing bacteria (October 2009) 183 Nr. Crt. Variant 1 V1 % mg/Kg Humus Indice N2 2.9 0.14 Data on the dinamics of some microbial groups... / Ovidius University Annals, Biology-Ecology Series 14: 181-184 (2010) 2 3 4 5 6 7 V2 V3 V4 V5 V6 M1 2.95 3.21 3.07 2.83 3.09 3.16 0.14 0.15 0.15 0.13 0.15 0.15 their activities by introducing large amounts of organic matter. 5. References [1] BERCA M, 2008 – Probleme de ecologia solului. Editura ceres, 2008: 43-63. [2] BERGEY’S, 1986 - Manual of Sistematic Bacteriology, vol. 2, Williams and Wilkins, Baltimore, USA, 4087: 1075-1079 [3] CLARK F, 1965 - Agar plate method for total microbial count. Method for Soil Analysis, vol.2: 1460-1465 Amercian Society for Agronomy, Madison, WL. [4] FLORENZANO G, 1983 - Fondamenti di microbiologia del rerreno, Reda Ed, Firenze, 630: 115-136. [5] PAPACOSTEA P, 1976 - Biologia solului, Ed. Ştiinţifică şi Enciclopedica, Bucureşti, 272: 81259. [6] PITT JL, 1991 - A Laboratory Guide to common Penicillium Species, USA, 184: 129-135. [7] TSUNEO WATANABE, 2001 - Pictorial Atlas of Soil and Seed Fungi, Morphologies of Cultured Fungi and Key to Species – Second edition, CRC Press, 504: 230-236. [8] DUMITRU M, TOTI M, VOICULESCU A-R, 2005 – Decontaminarea solurilor poluate cu compuşi organici, Ed. Sitech, Bucureşti, 364: 262-266. Table 2. Agrochemical analysis conducted after 6 months (May 2010) Nr. Crt. Variant % mg/Kg Humus Indice N2 1 2 3 4 5 6 7 V1 V2 V3 V4 V5 V6 M1 3.07 3.09 3.36 3.12 3.07 3.12 3.24 0.15 0.15 0.17 0.15 0.15 0.15 0.16 Further information relative determination of humus were not noted substantial increases for the period under review (Table 1, Table 2), which is understandable due to the short observation time insufficient to identify significant changes in the humus content. 4. Conclusions Dynamics of the total number of heterotrophic bacteria presented significant changes after application of amendments. The most significant increase occurred in the variant enriched with manure, trend was also observed in the case of variant VI in which soil was treated with Biovin. The total number of nitrogen fixing bacteria showed a spectacular increase after six months of amendments application, effect that can be attributed only in part as a result of fertilizer. Results of soil chemical and microbiological analysis reveal a low contribution of microorganisms to the improvement of the soil fertility and microbial biodiversity. To improve the biological quality of soil it is necessary to increase the biomass of microorganisms in the soil by adding bacteria, and 184 Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 THE AGRICULTURAL POTENTIAL OF PHOSPHOGYPSUM WASTE PILES Lucian MATEI Pescarusului Street, Bl CP1, Sc B, Ap 18, Navodari, Constanţa County, Romania, e-mail: mat_lucian@yahoo.com __________________________________________________________________________________________ Abstract: The cultivation of Salix sp. on the phosphogypsum waste piles started from the wish to discover a cheap, efficient, and ecological covering method. For this purpose, Salix alba and Salix fragilis cuttings were used, as they were collected from an area adjacent to the town of Navodari. Some Salix fragilis cuttings were collected from the trees that grew spontaneously on the waste piles. The species Salix alba is newly introduced in the ecosystem of the phosphogypsum waste pile. The species of the genus Salix are dioicous. As they are not fertile, S. alba and S. fragilis are often crossbred in nature, creating hybrids, the most popular being S. x rubens. The large number of hybrids of the genus Salix offers them increased capacity to adapt and exist in the most various environmental conditions. The purpose of the project is to identify a species or a hybrid that, given the life conditions on the phosphogypsum waste pile, should offer a considerable quantity of wood mass per ha in order to collect and exploit it as solid fuel. Keywords: Salix, phosphogypsum waste piles, ecological reconstruction, phytoreparation __________________________________________________________________________________________ 1. Introduction The idea of cultivating Salix sp. on the phosphogypsum waste piles started from the wish to discover a cheap, efficient, and ecological covering method. The phosphogypsum waste pile number 3, which belongs to the S.C. Fertilchim – Marway S.A. company, is the result of massive accumulations of phosphogypsum obtained by the wet method of making phosphorus fertilizers. The waste pile is rectangular and has a surface of approximately 21 ha. In 1996, it was removed from the technological flux and a poor vegetation settled spontaneously on its surface over the next few years. A study regarding flora, accomplished in 2009, identified 35 species of plants [1]. The dominant species is Puccinellia distans, a grass that prefers salty soils (Poaceae) [2]. Apart from this dominant species, waste pile number 3 also displays a mixture of various species in terms of preference for the environmental conditions. Thus, xerophile species such as Tamarix ramosissima live together with hygrophile species such as Salix fragilis and Salix matsudana. We can also encounter ISSN-1453-1267 spontaneous Salix caprea (mesophile) on the phosphogypsum waste pile. The species of the genus Salix are dioicous, the sexes being separate: the trees bear male or female flowers. Some species of the genus Salix are interfertile. Different varieties of Salix alba and Salix fragilis crossbreed frequently in nature giving birth to different hybrids, among which the most common is Salix x rubens. The overlapping of the morphological features of the two species increases the degree of difficulty in the correct determination of the species [3]. Both the morphological studies and the genetic investigations realized on the S. alba – S. x rubens – S. fragilis complex indicate its division into two main groups. A group is made up of Salix alba and Salix x rubens, while the second group is made up of S. fragilis si S. x rubens var. basfordiana [4]. This division of the complex into the two groups concords with previous research (Triest et al., 1998, 2000), quoted by [4], who analyzed the isoenzymes and RAPD (Random Amplified Polymorphic DNA). As a result of these tests, the S. alba – S. x rubens – S. fragilis complex was divided into two groups: “S. alba-like” and “S. fragilis-like”. © 2010 Ovidius University Press The agricultural potential... / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010) The great variety of hybrids existing in nature prevents sometimes the exact identification of the species of the genus Salix only by morphological 0.7 meters and eight rows (four with S. alba and four with S. fragilis) were planted at a depth of one meter. The collection of cuttings occurred between 10-20 March 2010, while features. It is possible that the specimens identified on the phosphogypsum waste pile as belonging to the species S. fragilis, might be hybrids that inherited from the genitors the capacity to live on a salty substrate which lacks organic matter but has high humidity. Unfortunately, the lack of financial means prevented the accomplishment of ADNcp (ADN chloroplastic) analyses that allow the precise identification of the species or supposed hybrids used within the experimental project for the setting up of a willow culture. The large number of hybrids of species of the genus Salix offers them increased capacities to adapt to the most diverse environmental conditions. Starting from this theory, the experimental culture using species of the genus Salix on the phosphogypsum waste pile seeks to identify a species or a hybrid that, in the living conditions of the waste pile, should provide the most considerable quantity of wood mass. I mention that the species Salix alba is newly introduced in the ecosystem of the phosphogypsum waste pile with the purpose of monitoring the production of biomass reported per surface unit. their planting took place between 16-27 March 2010. Some of the cuttings (22 pieces) were collected from S. fragilis grown spontaneously on the phosphogypsum pile, while the others (88 pieces) – 44 S. alba and 44 S. fragilis – were collected from the area adjacent to the town of Navodari. The age of the cuttings is between one and three years, while their sizes vary between one and 2.5 meters. In order to verify the influence of the microclimate effect, five rows of cuttings were planted in phosphogypsum ditches. The depth of the ditch was approximately 0.4 meters, while the width was 0.3 meters. The planting depth in four of the five ditches was measured to be one meter, taking the phosphogypsum surface as marker and not one meter, taking the bottom of the ditch as marker. In the case of the fifth ditch, the planting depth is 0.7 meters and it was measured the same as in the previous rows. The planting method is presented in Figure 1. 2. Material and Methods The experimental culture of Salix sp. on phosphogypsum waste pile no. 3 belonging to the S.C. Fertilchim – Marway S.A. company was set up on a vegetation-free surface of 540 square meters. For this purpose, Salix alba and Salix fragilis cuttings were used. They were collected from an area adjacent to the town of Navodari. Some Salix fragilis cuttings were collected from the trees that grew spontaneously on the waste piles. This surface was planted with 110 cuttings belonging to the species S. alba (44 pieces) and S. fragilis (6 pieces). The cuttings were planted on parallel rows with a length of 20 m. The distance between two successive rows is three meters, while the distance between the cuttings on the same row is two meters. The number of cuttings on a row is eleven. The planting depth is between 0.7 and one meter. Thus, two rows with S. fragilis were planted at Fig.1. The way in which the cuttings were planted The other five rows that make up the witness area for the study of the microclimate influence were planted directly on the phosphogypsum surface, without digging ditches. The planting depth in the case of four out of five rows is one meter, while a row was planted at 0.7 meters. 186 Matei Lucian / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010) No preparation or maintenance works were done before and after planting the newly established culture (e.g. fertilization, irrigation, etc). no samples for the humidity test were collected in December 2009 because it rained on the day scheduled for the sample collection (December 12, 2009). The graph in Fig. 2 presents the variation of humidity depending on the time and depth for the sample collection. 3. Results and Discussions The monitoring of the growth and development of the two willow species used to set up the experimental culture on the phosphogypsum waste pile, namely S. alba and S. fragilis, led to surprising results. Thus, on April 8, 2010, twelve days after the planting, it was observed that 107 out of the 110 planted cuttings took root and sprouts had developed on them, while the first two-three leaves had already emerged in a small number of these cuttings. Three of the 110 cuttings did not take root and dried out. Two of these belong to S. fragilis and one to S. alba. On April 18, 2010, the sprouts on the 107 cuttings opened and the first leaves emerged. Some of them even grew three-four cm long shoots. On May 20, 2010, it was observed that of the 107 cuttings that bore sprouts and shoots only 81 developed normally, with 5-20 cm long shoots. The other 26 were stagnating. The same situation was encountered on June 1, 2010, with the specification that the 81 cuttings with normal development had 10-35 cm long shoots and some of the 26 stagnating cuttings began to dry. We must mention that on the two rows (one with ditch for the verification of the microclimate influence and one without ditch, as witness), where the planting depth was 0.7 meters, only S. fragilis was used. These two rows registered the highest number of stagnating cuttings about to get dry. The conclusion is thus that the planting depth is very important, the greater the depth, the more chances the cuttings have to take root and develop normally. By taking phosphogypsum samples from various depths and performing humidity analyses, it was observed that humidity increases directly proportionally with the depth of the sample. Moreover, by analyzing samples from the surface of the phosphogypsum (0-5 cm) and those from the bottom of the ditches (40 cm), it was noticed over a period of several months that the samples from greater depths contained more water even though the months when the sample was collected were poor in precipitations. We specify that Fig. 2. The variation of humidity depending on the time and depth of the sample collection This situation explains to a large extent the surprising presence of certain xerophyte species alongside hygrophyte ones on the phosphogypsum waste pile. In order to make a correct estimation of the rooting and normal development of the cuttings depending on species, we will only take into account the eight rows on which the cuttings were planted at a depth of one meter (four with ditch for the verification of the microclimate effect and four without ditch, as witness). These eight rows include four rows planted with S. alba and four rows planted with S. fragilis. The total number of cuttings on these eight rows is 88, of which S. alba – 44, and S. fragilis – 44. Of the 44 S. alba cuttings planted on four of the eight rows, 38 develop normally, while of the 44 S. fragilis cuttings planted on four of the eight rows, only 32 develop normally. Though it is premature to draw pertinent conclusions, we can say that S. alba seems to be better adapted to the conditions of the phosphogypsum waste pile, considering that its percentage of rooting and development is 86.36%, compared to S. fragilis whose percentage is 72.72%. Taking these results 187 The agricultural potential... / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010) into account, it is surprising why S. alba did not emerge spontaneously on the phosphogypsum waste pile, considering that we identified a female specimen from this species located at less than one km from the pile. This observation represents another argument in favor of the hypothesis that the species between 4.7 and 6.56. These results, corroborated with the fact that the quoted species prefer a slightly acid pH, make the phosphogypsum waste pile a favorable environment for the setting up of a willow culture. S. fragilis and S. matsudana existing on the phosphogypsum waste pile occurred by vegetative reproduction and not sexual one (from seeds). In regards to the lower rooting and development degree, it is probable that the age of the cuttings used for planting had an important role in this aspect. Thus, all the S. alba cuttings were young (under one year old), while those of S. fragilis were older (between two and three years old). The phosphogypsum on waste pile no. 3, belonging to the S.C. Fertilchim – Marway S.A. company, contains 90% calcium sulfate or gypsum hydrated with water molecules (CaSO 4 x2H 2 O), alongside which we can encounter phosphorus pentoxide (P 2 O 5 ), traces of fluorhidric acid (HF), silicate (SiO 2 ) and high concentrations of heavy metals [5]. An analysis bulleting released by the Constanta County Office for Agronomical Studies and Pedology on May 6, 2009 attests to the fact that the analyzed phosphogypsum contains no organic matter (humus), nor nitrogen (N). The nutrients contained by the phosphogypsum are potassium (K) in very low quantity and a higher amount of phosphorus (P), a fact also demonstrated by the analyses accomplished by the method of extraction with lactate acetate (A.L.), which were realized in the Pedology Laboratory of the Faculty for Natural and Agricultural Sciences within “Ovidius” University. Even though it is hard to believe that there are species that can develop normally on a 100% mineral substrate, these four willow species (S. fragilis, S. matsudana and S caprea – spontaneous, and S. alba – introduced artificially) contradict this statement. Another factor that makes possible the normal development of these species directly on phosphogypsum is their resistance in conditions of high soil salinity. Thus, S. fragilis, S. matsudana and S. seringeana tolerate high salinity values [6], while S. alba is “the most tolerant of all willow species to brackish water” [7]. In parallel, pH analyses were accomplished on samples collected from depths between 5 and 100 cm which displayed pH values As the graph in Fig. 3 shows, no correlation can be made between pH value and the depth of sample collection. Fig. 3. The pH variation depending on the time and depth of the sample collection The only plausible explanation regarding this random distribution of the pH values in the phosphogypsum deposit could be that at the moment when the phosphogypsum suspension was neutralized, the milk of lime used did not always have the proper concentration. Willow is one of the well studied plants in order to use it in the phytoreparation processes, as it has a high capacity to accumulate heavy metals and it is easy to cultivate (Tremela et al. 1997; Pulford and Watson 2003) quoted by [8]. By concentrating important quantities of heavy metals in the shoots that will be collected every year, the willows will accomplish a depollution of the phosphogypsum and this will be a first step towards its transformation into organic-mineral fertilizer when the willow plantation will be eliminated. The strong bioaccumulation phenomenon in the species of the genus Salix will be favored by the slightly acid pH and will accelerate the cleansing of the phosphogypsum [9]. 188 Matei Lucian / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010) The harvesting should be done between November-February, after the leaves fall from the shoots. For harvesting, Claas Jaguar 880 GBE 1 or Claas Jaguar combines fitted with a HS2 harvesting head will be used. This type of combines transform the harvested shoots into a hash [10] that is left to dry and is then used in thermal power stations especially adapted for this solid fuel. The hash can be no precipitations. The optimum planting depth is 100 cm. The age of the cuttings has a very important role in the rooting process and their normal development. Thus, in the case of the one-year-old cuttings, the success percentage was higher. Over the entire surface of the waste pile, between 0 and 100 cm, the distribution of the pH values is purely random and they range between a minimum of 4.7 and a maximum of 6.56. This fact transformed into pellets used in regular thermal power stations as solid fuel. It is premature to speak about the role of the microclimate. By observing the phenotypical development of the willows planted in ditches and by comparing them to the willows planted directly on phosphogypsum, no major differences were noticed in regard to the length of the shoots and the plant vigor. In the case of the number of cuttings that display normal development, there are however small differences. Thus, in the case of S. fragilis, on the rows planted in ditches, there is a number of 17 cuttings that develop normally, compared to only 15 cuttings with normal development that were planted directly on phosphogypsum (witness area). In the case of S. alba on the rows planted in ditches, 20 cuttings develop normally, compared to only 17 cuttings with normal development planted directly on the phosphogypsum (witness area). It was noticed that the microclimate effect created by the ditches into phosphogypsum have a very important role in the case of seed germination and development of the annual herbaceous species. Thus, a few months after the digging of the ditches, a large number of herbaceous plants developed on their bottom. These plants germinated from seeds brought by the wind, mostly belonging to the dominant species in the waste pile phytocoenosis, Puccinellia distans. favors the development of species of the genus Salix, which prefer a substrate with slightly acid pH. The total lack of organic matter and of nitrogen from the substrate does not prevent the species from the genus Salix to develop normally, but it is possible to lead to a lower quantity of wood mass per surface unit. Even though within the experimental culture there was a larger number of cuttings of the species S. alba with normal development, it is premature to say that this species is better adapted to the environmental conditions than S. fragilis, a spontaneous species in the phosphogypsum waste pile ecosystem. It is also premature to draw a conclusion about the influence of the microclimate generated by the ditches into phosphogypsum on the development of the S. alba and S. fragilis cuttings. However, it was noticed that the microclimate generated by the ditches has a beneficial influence on the species of annual plants. Thus, in an interval of three months, a large number of herbaceous plants emerged on the bottom of the ditches, most of them belonging to Puccinellia distans, a dominant species in the phytocoenosis of the phosphogypsum deposit. The advantages of a culture with species belonging to the genus Salix on the phosphogypsum waste piles are multiple: - To obtain ecological fuel – the carbon eliminated by burning represents the carbon - Stored previously by photosynthesis, so no extra amounts of carbon are released into the atmosphere; - To use fields otherwise improper for other cultures and transform thus losses into profit; - The development of the root system and of the willow shoots will prevent wind erosion; 4. Conclusions In order to set up a willow culture, it is best to harvest and plant the cuttings between February 15 – March 15. The planting depth is very important. It was observed that at depths exceeding 40 cm, phosphogypsum always displays humidity over 25% even if the sample was collected after a period with 189 The agricultural potential... / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010) - - Taking into account the fact that the harvest of shoots takes place between November and February, the annual leaf litter on the phosphogypsum surface will accelerate the process of soil formation; By the phenomenon of bioaccumulation, the trees (shrubs) from the plantation will concentrate into their own structures important quantities of heavy metals that will be removed annually by cutting the shoots and decreasing thus the polluting content of the phosphogypsum; [5]. www.containment.fsu.edu/cd/content/pdf/466.pd f. Vegetative cover for phosphogypsum dumps: A Romanian field study. [6]. CROUCH R.J, Honeyman M.N., 1986, 'The relative salt tolerance of willow cuttings.' Journal of Soil Conservation, vol 42 (2), p. 103-104. [7]. ZALLAR S. Botanical Characteristics of the Willows, Soil Conservation Authority, Kew. [8]. www.sci.uszeged.hu/ABS/2006/Acta%20HP/50 37.pdf. Change of root and rhizosphere characters of willow (Salix sp) induced by high heavy metal pollution. [9]. www.umass.edu/.../Phytoremediation%20PDF /PhytoLitReview.pdf. Phytoremediation literature review. - The species of the genus Salix, fond of humidity, will retain part of the water resulted from precipitations, reducing drastically the levigation phenomenon and the draining of the phosphorus into the ground water; - The photosynthesis and evapo-transpiration generated by the willows will improve air quality and the local microclimate during the warm season. The main disadvantage is the fact that, being a monoculture, it will be more vulnerable to pests. Another possible disadvantage is that, because the cuttings are not planted like in a culture on swampy or irrigated land (the same density per square meter), the quantity of wood mass per ha can be reduced. [10]. www.bioeng.ca/pdfs/meetingpapers/2005/CSAE%20papers/05-080.pdf. Cutting, bundling and chipping shortrotation willow. 5. References [1]. SÂRBU I., Stefan N., Ivănescu Lăcrămioara, Mânzu C., 2001. Flora ilustrată a plantelor vasculare din estul României, Determinator, vol. I şi II, Editura Universităţii „Alexandru Ioan Cuza”, Iaşi. [2]. GOMOIU M.-T., Skolka M., 2001. Ecologie Metodologii pentru studii ecologice, Ovidius University Press, Constanţa. [3]. SKVORTSOVA. K., 1999. Willows of Russia and adjacent countries. Taxonomical and geographical review. Univ. Joensuu Fac. Mathem. and Natru. Sci. Rept. Ser. 39. 307 pp. www.bfafh.de/inst2/sg-pdf/52_3-4_148.pdf. [4]. Diversity of dte willow complex Salix alba – S x. rubens – S. Fragilis 190

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